Klotho beta

ABSTRACT

The invention concerns uses of anti-KLβ agents, and detection of KLβ and/or FGF19 and/or FGFR4.

RELATED APPLICATIONS

This is a continuation application of U.S. patent application Ser. No.12/594,443, filed Oct. 2, 2009 which is a National Stage ofInternational Patent Application Serial No. PCT/US2008/059032, filedApr. 1, 2008, which claims priority to U.S. Patent Application Ser. No.60/909,699 filed on Apr. 2, 2007 and U.S. Patent Application Ser. No.60/916,187 filed on May 4, 2007, both of which are incorporated hereinby reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 25, 2015, isnamed P2477R1C3US_Sequence_Listing.txt and is 37,496 bytes in size.

FIELD OF THE INVENTION

The present invention relates generally to the fields of molecularbiology. More specifically, the invention concerns uses of anti-KLβagents, and detection of KLβ and/or FGF19 and/or FGFR4.

BACKGROUND OF THE INVENTION

Klotho beta (“KLβ”, “KLB” or “beta klotho”) is a 130-kDa type 1transmembrane protein with a short (29 amino acids) intracellular domainthat has no predicted kinase activity (Ito et al., Mech. Dev. 98 (2000)115-119). KLβ has two extracellular glycosidase domains that lack acharacteristic glutamic acid residue essential for enzymatic activity.Klb-deficient mice (Klb−/−mice) have increased CYP7A1 expression anddecreased gallbladder size, indicating that Klb−/−mice can no longersuppress bile acid synthesis (Inagaki, T et al (2005) Cell Metab2:217-25). KLβ is predominantly expressed in liver and pancreas. Id.Disruption of the gene encoding KLβ in mice results in marked increasesin mRNA levels of cholesterol 7alpha-hydroxylase (CYP7A1), the first andrate-limiting enzyme in the bile acid biosynthetic pathway. Ito et al(2005) J Clin Invest 115(8):2202-2208; Arrese et al (2006) Hepatology43(1):191-193; Moschetta and Kliewer (2005) J Clin Invest 115(8):2075-2077.

The fibroblast growth factor (FGF) family is composed of 22 structurallyrelated polypeptides that bind to 4 receptor tyrosine kinases (FGFR1-4)and one kinase deficient receptor (FGFR5) (Eswarakumar et al (2005)Cytokine Growth Factor Rev 16, 139-149; Ornitz et al (2001) Genome Biol2, REVIEWS3005; Sleeman et al (2001) Gene 271, 171-182). FGFs'interaction with FGFR1-4 results in receptor homodimerization andautophosphorylation, recruitment of cytosolic adaptors such as FRS2 andinitiation of multiple signaling pathways (Powers et al (2000) EndocrRelat Cancer 7, 165-197; Schlessinger, J. (2004) Science 306,1506-1507).

FGFs and FGFRs play important roles in development and tissue repair byregulating cell proliferation, migration, chemotaxis, differentiation,morphogenesis and angiogenesis (Ornitz et al (2001) Genome Biol 2,REVIEWS3005; Auguste et al (2003) Cell Tissue Res 314, 157-166; Steilinget al (2003) Curr Opin Biotechnol 14, 533-537). Several FGFs and FGFRsare associated with the pathogenesis of breast, prostate, cervix,stomach and colon cancers (Jeffers et al (2002) Expert Opin Ther Targets6, 469-482; Mattila et al. (2001) Oncogene 20, 2791-2804; Ruohola et al.(2001) Cancer Res 61, 4229-4237; Marsh et al (1999) Oncogene 18,1053-1060; Shimokawa et al (2003) Cancer Res 63, 6116-6120; Jang (2001)Cancer Res 61, 3541-3543; Cappellen (1999) Nat Genet 23, 18-20;Gowardhan (2005) Br J Cancer 92, 320-327).

FGF19 is a member of the most distant of the seven subfamilies of theFGFs. FGF19 is a high affinity ligand of FGFR4 (Xie et al (1999)Cytokine 11:729-735). FGF19 is normally secreted by the biliary andintestinal epithelium. FGF19 plays a role in cholesterol homeostasis byrepressing hepatic expression of cholesterol-7-α-hydroxylase 1 (Cyp7α1),the rate-limiting enzyme for cholesterol and bile acid synthesis(Gutierrez et al (2006) Arterioscler Thromb Vasc Biol 26, 301-306; Yu etal (2000) J Biol Chem 275, 15482-15489; Holt, J A, et al. (2003) GenesDev 17(130):158). FGF19 ectopic expression in a transgenic mouse modelincreases hepatocyte proliferation, promotes hepatocellular dysplasiaand results in neoplasia by 10 months of age (Nicholes et al. (2002). AmJ Pathol 160, 2295-2307). The mechanism of FGF19 induced hepatocellularcarcinoma is thought to involve FGFR4 interaction. FGF19 overexpressionin tumor tissues is described in co-owned co-pending U.S. patentapplication Ser. No. 11/673,411 (filed Feb. 9, 2007). Transgenic miceectopically expressing FGF19 weigh less than their wild-typelittermates, due in part to decrease in white adipose tissue. Tomlinson,E et al. (2002) Endocrinology 143:1741-1747. Although FGF19 transgenicmice have increased food intake, they also have a higher metabolic ratethat is independent of increases in leptin, IGF-1, growth hormone, orthyroid hormone levels. Similarly, treatment with FGF-19 increasedmetabolic rate and reverses dietary and leptin-deficient diabetes. FGF19administration improved glucose tolerance and decreased serum insulin,leptin, cholesterol and triglycerides. Fu et al (2004) 145:2594-2603.Administration of recombinant FGF19 to ob/ob mice, or crossing the FGF19transgenic mice onto the ob/ob background resulted in mice that weighedless and had lower serum glucose levels and improved glucose sensitivitycompared to ob/ob mice. Id. FGF-19 is also described in, for example,Harmer et al (2004) Biochemistry 43:629-640.

FGFR4 expression is widely distributed and was reported in developingskeletal muscles, liver, lung, pancreas, adrenal, kidney and brain (Kanet al. (1999) J Biol Chem 274, 15947-15952; Nicholes et al. (2002). Am JPathol 160, 2295-2307; Ozawa et al. (1996) Brain Res Mol Brain Res 41,279-288; Stark et al (1991) Development 113, 641-651). FGFR4amplification was reported in mammary and ovarian adenocarcinomas(Jaakkola et al (1993) Int J Cancer 54, 378-382). FGFR4 mutation andtruncation were correlated with the malignancy and in some cases theprognosis of prostate and lung adenocarcinomas, head and neck squamouscell carcinoma, soft tissue sarcoma, astrocytoma and pituitary adenomas(Jaakkola et al (1993) Int J Cancer 54, 378-382; Morimoto (2003) Cancer98, 2245-2250; Qian (2004) J Clin Endocrinol Metab 89, 1904-1911;Spinola et al. (2005) J Clin Oncol 23, 7307-7311; Streit et al (2004)Int J Cancer 111, 213-217; Wang (1994) Mol Cell Biol 14, 181-188; Yamada(2002) Neurol Res 24, 244-248). FGFR4 overexpression in tumor tissues isdescribed in WO2007/13693.

It is clear that there continues to be a need for agents that haveclinical attributes that are optimal for development as therapeuticagents. The invention described herein meets this need and providesother benefits.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

It is demonstrated herein that FGF19 requires KLβ for binding to FGFR4,FGFR4 downstream signaling and down-stream gene modulation. Thus, it isshown that KLβ and its interaction with FGFR can be a unique andadvantageous target for greater fine-tuning in designing prophylaticand/or therapeutic approaches against pathological conditions associatedwith abnormal or unwanted signaling of the FGF/FGFR pathway. Thus, theinvention provides methods, compositions, kits and articles ofmanufacture for identifying and using substances that are capable ofmodulating the FGF/FGFR pathways through modulation of KLβ binding toFGFR and modulation of KLβ binding to FGFs, and for modulation ofbiological/physiological activities associated with FGF/FGFR signaling.KLβ presents as an important and advantageous therapeutic target, andthe invention also provides compositions and methods based on bindingKLβ. KLβ binding agents, as described herein, provide importanttherapeutic and diagnostic agents for use in targeting pathologicalconditions associated with expression and/or activity of theKLβ-FGF-FGFR pathways.

In one aspect, the invention provides methods, compositions, kits andarticles of manufacture related to KLβ binding and detection of KLβand/or FGF19 and/or FGFR4 binding.

In one aspect, the invention provides methods and compositions usefulfor modulating disease states associated with expression and/or activityof KLβ, such as increased expression and/or activity or undesiredexpression and/or activity, said methods comprising administration of aneffective dose of a KLβ antagonist (such as an anti-KLβ antibody) to anindividual in need of such treatment.

In one aspect, the invention provides methods for treating a tumor,cancer, or cell proliferative disorder comprising administering aneffective amount of a KLβ antagonist (such as an anti-KLβ antibody) toan individual in need of such treatment. In some embodiments, the tumor,cancer, or cell proliferative disorder is hepatocellular carcinoma,pancreatic cancer, non-small cell lung cancer, breast cancer, orcolorectal cancer.

In one aspect, the invention provides methods for killing a cell (suchas a cancer or tumor cell), the methods comprising administering aneffective amount of a KLβ antagonist (such as an anti-KLβ antibody) toan individual in need of such treatment. In some embodiments, the cellis a hepatocellular carcinoma cell or a pancreatic cancer cell. In someembodiments, the cell is a liver or pancreatic cell.

In one aspect, the invention provides methods for reducing, inhibiting,blocking, or preventing growth of a tumor or cancer, the methodscomprising administering an effective amount of a KLβ antagonist (suchas an anti-KLβ antibody) to an individual in need of such treatment. Insome embodiments, the tumor, cancer, or cell proliferative disorder ishepatocellular carcinoma, pancreatic cancer, non-small cell lung cancer,breast cancer, or colorectal cancer.

In one aspect, the invention provides methods for treating and/orpreventing a liver disorder, the methods comprising administering aneffective amount of a KLβ antagonist (such as an anti-KLβ antibody) toan individual in need of such treatment. In some embodiments, the liverdisorder is cirrhosis.

In one aspect, the invention provides methods for treating a wastingdisorder comprising administering an effective amount of a KLβantagonist (such as an anti-KLβ antibody) to an individual in need ofsuch treatment. In some embodiments, the individual has a tumor, acancer, and/or a cell proliferative disorder.

In one aspect, the invention provides methods for treating hypoglycemiacomprising administering an effective amount of a KLβ antagonist (suchas an anti-KLβ antibody) to an individual in need of such treatment.

In one aspect, the invention provides methods for treating cholestasisor dysregulation of bile acid metabolism comprising administering aneffective amount of a KLβ antagonist (such as an anti-KLβ antibody) toan individual in need of such treatment.

In one aspect, the invention provides methods for treating obesity or anobesity-related condition comprising administration of an effective doseof a KLβ agonist to an individual in need of such treatment. In someembodiments, the obesity-related condition is diabetes mellitus,cardiovascular disease, insulin resistance, hypertension,hypercholesterolemia, thromboembolic disease (such as stroke),atherosclerosis, dyslipidemia (for example, high total cholesterol orhigh triglyceride levels), osteoarthritis, gallbladder disease,osteoarthritis, and sleep apnea and other respiratory disorders.

In one aspect, the invention provides methods for inducing an increasein insulin sensitivity comprising administration of an effective dose ofa KLβ agonist to an individual in need of such treatment.

In one aspect, the invention provides methods for reducing total bodymass comprising administration of an effective dose of a KLβ agonist toan individual in need of such treatment.

In one aspect, the invention provides methods for treating hyperglycemiacomprising administration of an effective dose of a KLβ agonist to anindividual in need of such treatment.

In one aspect, the invention provides methods for reducing at least oneof triglyceride and free fatty acid levels comprising administration ofan effective dose of a KLβ agonist to an individual in need of suchtreatment.

Methods of the invention can be used to affect any suitable pathologicalstate. Exemplary disorders are described herein.

In one embodiment, a cell that is targeted in a method of the inventionis a cancer cell. For example, a cancer cell can be one selected fromthe group consisting of a hepatocellular carcinoma cell or a pancreaticcancer cell. In one embodiment, a cell that is targeted in a method ofthe invention is a hyperproliferative and/or hyperplastic cell. In oneembodiment, a cell that is targeted in a method of the invention is adysplastic cell. In yet another embodiment, a cell that is targeted in amethod of the invention is a metastatic cell. In one embodiment, thecell that is targeted is a cirrhotic liver cell.

Methods of the invention can further comprise additional treatmentsteps. For example, in one embodiment, a method further comprises a stepwherein a targeted cell and/or tissue (for e.g., a cancer cell) isexposed to radiation treatment or a chemotherapeutic agent.

KLβ antagonists and agonists are known in the art and some are describedand exemplified herein. In some embodiments, the KLβ antagonist is amolecule which binds to KLβ and neutralizes, blocks, inhibits,abrogates, reduces or interferes with one or more aspects ofKLβ-associated effect.

In some embodiments, the KLβ antagonist is an antibody. In someembodiments, the antibody is a monoclonal antibody. In some embodiments,the antibody is a polyclonal antibody. In some embodiments, the antibodyis selected from the group consisting of a chimeric antibody, anaffinity matured antibody, a humanized antibody, and a human antibody.In some embodiments, the antibody is an antibody fragment. In someembodiments, the antibody is a Fab, Fab′, Fab′-SH, F(ab′)₂, or scFv.

In one embodiment, the antibody is a chimeric antibody, for example, anantibody comprising antigen binding sequences from a non-human donorgrafted to a heterologous non-human, human or humanized sequence (e.g.,framework and/or constant domain sequences). In one embodiment, thenon-human donor is a mouse. In one embodiment, an antigen bindingsequence is synthetic, e.g. obtained by mutagenesis (e.g., phage displayscreening, etc.). In one embodiment, a chimeric antibody has murine Vregions and human C region. In one embodiment, the murine light chain Vregion is fused to a human kappa light chain. In one embodiment, themurine heavy chain V region is fused to a human IgG1 C region.

Humanized antibodies useful in methods of the invention include thosethat have amino acid substitutions in the FR and affinity maturationvariants with changes in the grafted CDRs. The substituted amino acidsin the CDR or FR are not limited to those present in the donor orrecipient antibody. In other embodiments, the antibodies furthercomprise changes in amino acid residues in the Fc region that lead toimproved effector function including enhanced CDC and/or ADCC functionand B-cell killing. Other antibodies include those having specificchanges that improve stability. In other embodiments, the usefulantibodies comprise changes in amino acid residues in the Fc region thatlead to decreased effector function, e.g. decreased CDC and/or ADCCfunction and/or decreased B-cell killing. In some embodiments, theantibodies are characterized by decreased binding (such as absence ofbinding) to human complement factor C1q and/or human Fc receptor onnatural killer (NK) cells. In some embodiments, the antibodies arecharacterized by decreased binding (such as the absence of binding) tohuman FcγRI, FcγRIIA, and/or FcγRIIIA. In some embodiments, the antibodyis of the IgG class (e.g., IgG1 or IgG4) and comprises at least onemutation in E233, L234, L235, G236, D265, D270, N297, E318, K320, K322,A327, A330, P331 and/or P329 (numbering according to the EU index). Insome embodiments, the antibodies comprise the mutation L234A/L235A orD265A/N297A.

In one aspect, the KLβ antagonist is an anti-KLβ polypeptide comprisingany of the antigen binding sequences provided herein, wherein theanti-KLβ polypeptides specifically bind to KLβ.

In one aspect, the KLβ antagonist is an immunoconjugate (interchangeablytermed “antibody drug conjugate” or “ADC”) comprising an anti-KLβpolypeptide (such as an anti-KLβ antibody) conjugated to an agent, suchas a drug.

In one aspect, the KLβ antagonist is a KLβ siRNA. Examples of KLβ siRNAare described herein.

In some embodiments, the KLβ antagonist may modulate one or more aspectsof KLβ-associated effects, including but not limited to binding FGFR(e.g., FGFR4) (optionally in conjunction with heparin), binding FGF(e.g., FGF19) (optionally in conjunction with heparin), binding FGFR4and FGF19 (optionally in conjunction with heparin), promotingFGF19-mediated induction of cFos, Junb and/or Junc (in vitro or invivo), promoting FGFR4 and/or FGF19 downstream signaling (including butnot limited to FGFR phosphorylation, FRS2 phosphorylation, ERK1/2phosphorylation and Wnt pathway activation), and/or promotion of anybiologically relevant KLβ and/or FGFR and/or FGF biological pathway,and/or promotion of a tumor, cell proliferative disorder or a cancer;and/or promotion of a disorder associated with KLβ expression and/oractivity (such as increased KLβ expression and/or activity). In someembodiments, the antagonist binds (such as specifically binds to KLβ).In some embodiments, the antagonist binds to an FGFR (such as FGFR4)binding region of KLβ. In some embodiments, the antagonist binds to aFGF (e.g., FGF19) binding regions of KLβ. In some embodiments, theantagonist reduces, inhibits, and/or blocks KLβ activity in vivo and/orin vitro. In some embodiments, the antagonist competes for binding withFGFR4 (reduces and/or blocks FGFR4 binding to KLβ). In some embodiments,the antagonist competes for binding with FGF19 (reduces and/or blocksFGF19 binding to KLβ).

In another aspect, the invention supplies a composition comprising oneor more KLβ antagonist (such as an anti-KLβ antibody), and a carrier.This composition may further comprise a second medicament, wherein theKLβ antagonist is a first medicament. This second medicament, for cancertreatment, for example, may be another KLβ antagonist (such as ananti-KLβ antibody), chemotherapeutic agent, cytotoxic agent,anti-angiogenic agent, immunosuppressive agent, prodrug, cytokine,cytokine antagonist, cytotoxic radiotherapy, corticosteroid, anti-emeticcancer vaccine, analgesic, anti-vascular agent, or growth-inhibitoryagent. In another embodiment, a second medicament is administered to thesubject in an effective amount, wherein the antibody is a firstmedicament. This second medicament is more than one medicament, and ispreferably another antibody, chemotherapeutic agent, cytotoxic agent,anti-angiogenic agent, immunosuppressive agent, prodrug, cytokine,cytokine antagonist, cytotoxic radiotherapy, corticosteroid,anti-emetic, cancer vaccine, analgesic, anti-vascular agent, orgrowth-inhibitory agent. More specific agents include, for example,irinotecan (CAMPTOSAR®), cetuximab (ERBITUX®), fulvestrant (FASLODEX®),vinorelbine (NAVELBINE®), EFG-receptor antagonists such as erlotinib(TARCEVA®) VEGF antagonists such as bevacizumab (AVASTIN®), vincristine(ONCOVIN®), inhibitors of mTor (a serine/threonine protein kinase) suchas rapamycin and CCI-779, and anti-HER1, HER2, ErbB, and/or EGFRantagonists such as trastuzumab (HERCEPTIN®), pertuzumab (OMNITARG™), orlapatinib, and other cytotoxic agents including chemotherapeutic agents.In some embodiments, the second medicament is an anti-estrogen drug suchas tamoxifen, fulvestrant, or an aromatase inhibitor, an antagonist tovascular endothelial growth factor (VEGF) or to ErbB or the Efbreceptor, or Her-1 or Her-2. In some embodiments, the second medicamentis tamoxifen, letrozole, exemestane, anastrozole, irinotecan, cetuximab,fulvestrant, vinorelbine, erlotinib, bevacizumab, vincristine, imatinib,sorafenib, lapatinib, or trastuzumab, and preferably, the secondmedicament is erlotinib, bevacizumab, or trastuzumab.

In one aspect, the invention provides an article of manufacturecomprising a container; and a composition contained within thecontainer, wherein the composition comprises one or more KLβ antagonist(such as an anti-KLβ antibody). In one embodiment, a compositioncomprising a KLβ antagonist further comprises a carrier, which in someembodiments is pharmaceutically acceptable. In one embodiment, anarticle of manufacture of the invention further comprises instructionsfor administering the composition (for e.g., an anti-KLβ antibody) to anindividual (such as instructions for any of the methods describedherein).

In one aspect, the invention provides a kit comprising a first containercomprising a composition comprising one or more anti-KLβ antagonist; anda second container comprising a buffer. This composition may furthercomprise a second medicament, wherein the KLβ antagonist is a firstmedicament. Exemplary second medicaments are described above andelsewhere herein. In one embodiment, the buffer is pharmaceuticallyacceptable. In one embodiment, a composition comprising an antibodyfurther comprises a carrier, which in some embodiments ispharmaceutically acceptable. In one embodiment, a kit further comprisesinstructions for administering the composition (for e.g., the antibody)to an individual.

In another aspect, the invention provides methods for detection of KLβ,the methods comprising detecting KLβ in a sample (such as a biologicalsample). The term “detection” as used herein includes qualitative and/orquantitative detection (measuring levels) with or without reference to acontrol. In some embodiment, the biological sample is from a patienthaving or suspected of having a tumor, cancer, and/or a cellproliferative disorder, such as hepatocellular carcinoma, pancreaticcancer, non-small cell lung cancer, breast cancer, or colorectal cancer.In some embodiments, the biological sample is from a tumor. In someembodiments, the biological sample expresses FGF (e.g., FGF19) and/orFGFR (e.g., FGFR4).

In another aspect, the invention provides methods for detecting adisorder associated with KLβ expression and/or activity, the methodscomprising detecting KLβ in a biological sample from an individual. Insome embodiments, the KLβ expression is increased expression or abnormalexpression. In some embodiments, the disorder is a tumor, cancer, and/ora cell proliferative disorder, such as hepatocellular carcinoma,pancreatic cancer, non-small cell lung cancer, breast cancer, orcolorectal cancer. In some embodiment, the biological sample is serum orof a tumor.

In another aspect, the invention provides methods for detecting adisorder associated with FGFR4 and KLβ expression and/or activity, themethods comprising detecting FGFR4 and KLβ in a biological sample froman individual. In some embodiments, the KLβ expression is increasedexpression or abnormal expression. In some embodiments, FGFR4 expressionis increased expression or abnormal expression. In some embodiments, thedisorder is hepatocellular carcinoma, pancreatic cancer, non-small celllung cancer, breast cancer, or colorectal cancer. In some embodiment,the biological sample is serum or of a tumor. In some embodiments,expression of FGFR4 is detected in a first biological sample, andexpression of KLβ is detected in a second biological sample.

In another aspect, the invention provides methods for detecting adisorder associated with FGF19 and KLβ expression and/or activity, themethods comprising detecting FGF19 and KLβ in a biological sample froman individual. In some embodiments, the KLβ expression is increasedexpression or abnormal expression. In some embodiments, FGF19 expressionis increased expression or abnormal expression. In some embodiments, thedisorder is a tumor, cancer, and/or a cell proliferative disorder, suchas hepatocellular carcinoma, pancreatic cancer, non-small cell lungcancer, breast cancer, or colorectal cancer. In some embodiment, thebiological sample is serum or of a tumor. In some embodiments,expression of FGF19 is detected in a first biological sample, andexpression of KLβ is detected in a second biological sample.

In another aspect, the invention provides methods for detecting adisorder associated with FGFR4, FGF19, and KLβ expression and/oractivity, the methods comprising detecting FGFR4, FGF19 and KLβ in abiological sample from an individual. In some embodiments, the KLβexpression is increased expression or abnormal expression. In someembodiments, FGFR4 expression is increased expression or abnormalexpression. In some embodiments, the disorder is a tumor, cancer, and/ora cell proliferative disorder, such as hepatocellular carcinoma,pancreatic cancer, non-small cell lung cancer, breast cancer, orcolorectal cancer. In some embodiment, the biological sample is serum orfrom a tumor. In some embodiments, expression of FGFR4 is detected in afirst biological sample, expression of FGF19 is detected in a secondbiological sample, and expression of KLβ is detected in a thirdbiological sample.

In another aspect, the invention provides methods for treating anindividual having or suspected of having a cancer, a tumor, and/or acell proliferative disorder or a liver disorder (such as cirrhosis) byadministering an effective amount of a KLβ antagonist (e.g., an anti-KLβantibody), wherein a biological sample of the cancer, tumor and/or celldisorder or liver disorder expresses (i) KLβ, (ii) KLβ and FGFR4, (iii)KLβ and FGF19, or (iv) KLβ, FGFR4 and FGF19. In some embodiments, thecancer, rumor and/or cell proliferative disorder or liver disorder ishepatocellular carcinoma, pancreatic cancer, non-small cell lung cancer,breast cancer, or colorectal cancer.

In another aspect, the invention provides methods for treating anindividual having or suspected of having a cancer, a tumor, and/or acell proliferative disorder or a liver disorder (such as cirrhosis) byadministering an effective amount of a FGF19 antagonist (e.g., ananti-FGF19 antibody), wherein a biological sample of the cancer, tumorand/or cell disorder or liver disorder expresses (i) KLβ, (ii) KLβ andFGFR4, (iii) KLβ and FGF19, or (iv) KLβ, FGFR4 and FGF19. In someembodiments, the cancer, rumor and/or cell proliferative disorder orliver disorder is hepatocellular carcinoma, pancreatic cancer, non-smallcell lung cancer, breast cancer, or colorectal cancer.

In another aspect, the invention provides methods for treating anindividual having or suspected of having a cancer, a tumor, and/or acell proliferative disorder or a liver disorder (such as cirrhosis) byadministering an effective amount of an FGFR4 antagonist (e.g., ananti-FGFR4 antibody), wherein a biological sample of the cancer, tumorand/or cell disorder or liver disorder expresses (i) KLβ, (ii) KLβ andFGFR4, (iii) KLβ and FGF19, or (iv) KLβ, FGFR4 and FGF19. In someembodiments, the cancer, rumor and/or cell proliferative disorder orliver disorder is hepatocellular carcinoma, pancreatic cancer, non-smallcell lung cancer, breast cancer, or colorectal cancer.

In another aspect, the invention provides methods for selectingtreatment for an individual, the methods comprising: (a) determining (i)KLβ expression, (ii) KLβ and FGF19 expression, (iii) KLβ and FGFR4expression, or (iv) KLβ, FGF19 and FGFR4 expression, if any, in anindividual's biological sample; and (b) subsequent to step (a),selecting treatment for the individual, wherein the selection oftreatment is based on the expression determined in step (a). In someembodiments, increased KLβ expression in the individual's biologicalsample relative to a reference value or control sample is determined. Insome embodiments, decreased KLβ expression in the individual'sbiological sample relative to a reference value or control sample isdetermined in the individual. In some embodiments, KLβ expression isdetermined and treatment with an anti-KLβ antibody is selected. In someembodiments, KLβ expression is determined and treatment with an FGF19antagonist (such as an anti-FGF19 antibody) is selected. In someembodiments, KLβ expression is determined and treatment with an FRFR4antagonist (such as an anti-FGFR4 antibody) is selected. FGFR4antagonists are known in the art. In some embodiments, the individualhas a tumor, cancer, and/or a cell proliferative disorder, such ashepatocellular carcinoma, pancreatic cancer, non-small cell lung cancer,breast cancer, or colorectal cancer.

In another aspect, the invention provides methods for treating anindividual having or suspected of having a cancer, a tumor, and/or acell proliferative disorder or a liver disorder (such as cirrhosis) byadministering an effective amount of an anti-KLβ antibody, furtherwherein (i) KLβ expression, (ii) KLβ and FGF19 expression, (iii) KLβ andFGFR4 expression, or (iv) KLβ, FGF19 and FGFR4 expression is determinedin the individual's biological sample before, during or afteradministration of an anti-KLβ antibody. In some embodiments, thebiological sample is of the cancer, tumor and/or cell proliferativedisorder. In some embodiments, the biological sample is serum. In someembodiments, KLβ over-expression is determined before, during and/orafter administration of an anti-KLβ antibody. In some embodiments, FGFR4expression is determined before, during and/or after administration ofan anti-KLβ antibody. Expression may be determined before; during;after; before and during; before and after; during and after; or before,during and after administration of an anti-KLβ antibody.

In another aspect, the invention provides methods for treating anindividual having or suspected of having a cancer, a tumor, and/or acell proliferative disorder or a liver disorder (such as cirrhosis) byadministering an effective amount of an anti-FGF19 antibody, furtherwherein (i) KLβ expression, (ii) KLβ and FGF19 expression, (iii) KLβ andFGFR4 expression, or (iv) KLβ, FGF19 and FGFR4 expression is determinedin the individual's biological sample before, during or afteradministration of an anti-FGF19 antibody. In some embodiments, thebiological sample is of the cancer, tumor and/or cell proliferativedisorder. In some embodiments, the biological sample is serum. In someembodiments, KLβ over-expression is determined before, during and/orafter administration of an anti-FGF19 antibody. In some embodiments,FGFR4 expression is determined before, during and/or afteradministration of an anti-FGF19 antibody. Expression may be determinedbefore; during; after; before and during; before and after; during andafter; or before, during and after administration of an anti-FGF19antibody. Anti-FGF19 antibodies and methods of treatment comprising useof an anti-FGF19 antibody are described in co-owned co-pending U.S.patent application Ser. No. 11/673,411 (filed Feb. 9, 2007), thecontents of which are hereby incorporated by reference.

In embodiments involving detection, expression of FGFR4 downstreammolecular signaling may be detected in addition to or as an alternativeto detection of FGFR4 expression. In some embodiments, detection ofFGFR4 downstream molecular signaling comprises one or more of detectionof phosphorylation of MAPK, FRS2 or ERK1/2 (or ERK1 and/or ERK2).

In some embodiments involving detection, expression of FGFR4 comprisesdetection of FGFR4 gene deletion, gene amplification and/or genemutation. In some embodiments involving detection, expression of KLβcomprises detection of KLβ gene deletion, gene amplification and/or genemutation. In some embodiments involving detection, expression of FGF19comprises detection of FGF19 gene deletion, gene amplification and/orgene mutation.

Some embodiments involving detection further comprise detection of Wntpathway activation. In some embodiments, detection of Wnt pathwayactivation comprises one or more of tyrosine phosphorylation ofβ-catenin, expression of Wnt target genes, β-catenin mutation, andE-cadherin binding to β-catenin. Detection of Wnt pathway activation isknown in the art, and some examples are described and exemplifiedherein.

Biological samples are described herein, e.g., in the definition ofBiological Sample. In some embodiment, the biological sample is serum orof a tumor.

In embodiments involving detection of KLβ and/or FGFR4 and/or FGF19expression, KLβ and/or FGFR4 and/or FGF19 polynucleotide expressionand/or KLβ and/or FGFR4 and/or FGF19 polypeptide expression may bedetected. In some embodiments involving detection of KLβ and/or FGFR4and/or FGF19 expression, KLβ and/or FGFR4 and/or FGF19 mRNA expressionis detected. In other embodiments, KLβ and/or FGFR4 and/or FGF19polypeptide expression is detected using an anti-KLβ agent and/or ananti-FGFR4 agent. In some embodiments, KLβ and/or FGFR4 and/or FGF19polypeptide expression is detected using an antibody. Any suitableantibody may be used for detection and/or diagnosis, includingmonoclonal and/or polyclonal antibodies, a human antibody, a chimericantibody, an affinity-matured antibody, a humanized antibody, and/or anantibody fragment. In some embodiments, an anti-KLβ antibody describedherein is used for detection. In some embodiments, KLβ and/or FGFR4and/or FGF19 polypeptide expression is detected usingimmunohistochemistry (IHC). In some embodiments, polypeptide expressionis scored at 2 or higher using an IHC.

In some embodiments involving detection of KLβ and/or FGFR4 and/or FGF19expression, presence and/or absence and/or level of KLβ and/or FGFR4and/or FGF19 expression may be detected. KLβ and/or FGFR4 and/or FGF19expression may be increased. It is understood that absence of KLβ and/orFGFR4 and/or FGF19 expression includes insignificant, or de minimuslevels. In some embodiments, target expression in the test biologicalsample is higher than that observed for a control biological sample (orcontrol or reference level of expression). In some embodiments, targetexpression is at least about 2-fold, 5-fold, 10-fold, 20-fold, 30-fold,40-fold, 50-fold, 75-fold, 100-fold, 150-fold higher, or higher in thetest biological sample than in the control biological sample. In someembodiments, target polypeptide expression is determined in animmunohistochemistry (“IHC”) assay to score at least 2 or higher forstaining intensity. In some embodiments, target polypeptide expressionis determined in an IHC assay to score at least 1 or higher, or at least3 or higher for staining intensity. In some embodiments, targetexpression in the test biological sample is lower than that observed fora control biological sample (or control expression level).

In one aspect, the invention provides methods of identifying a candidateinhibitor substance that inhibits KLβ binding to FGFR (e.g., FGFR4),said method comprising: (a) contacting a candidate substance with afirst sample comprising FGFR, FGF (e.g., FGF19) and KLβ, and (b)comparing amount of FGFR biological activity in the sample with amountof FGFR biological activity in a reference sample comprising similaramounts of KLβ, FGF and FGFR as the first sample but that has not beencontacted with said candidate substance, whereby a decrease in amount ofFGFR biological activity in the first sample compared to the referencesample indicates that the candidate substance is capable of inhibitingKLβ binding to FGFR.

In another aspect, the invention provides methods of identifying acandidate inhibitor substance that inhibits KLβ binding to FGFR (e.g.,FGFR4), said method comprising: (a) contacting a candidate substancewith a first sample comprising FGFR, FGF and KLβ, and (b) comparingamount of FGFR-KLβ complex in the sample with amount of FGFR-KLβ complexin a reference sample comprising similar amounts of KLβ, FGF and FGFR asthe first sample but that has not been contacted with said candidatesubstance, whereby a decrease in amount of FGFR-KLβ complex in the firstsample compared to the reference sample indicates that the candidatesubstance is capable of inhibiting KLβ binding to FGFR.

In another aspect, the invention provides methods of determining whethera candidate substance inhibits KLβ binding to FGFR (e.g., FGFR4), saidmethod comprising: (a) contacting a candidate substance with a firstsample comprising FGFR, FGF and KLβ, and (b) comparing amount of FGFRbiological activity in the sample with amount of FGFR biologicalactivity in a reference sample comprising similar amounts of KLβ, FGFand FGFR as the first sample but that has not been contacted with saidcandidate substance, whereby a decrease in amount of FGFR biologicalactivity in the first sample compared to the reference sample indicatesthat the candidate substance is capable of inhibiting KLβ binding toFGFR.

In another aspect, the invention provides methods of determining whethera candidate substance inhibits FGF binding to KLβ, said methodcomprising: (a) contacting a candidate substance with a first samplecomprising FGF, FGFR and KLβ, and (b) comparing amount of FGFRbiological activity in the sample with amount of FGFR biologicalactivity in a reference sample comprising similar amounts of FGF, FGFRand KLβ as the first sample but that has not been contacted with saidcandidate substance, whereby a decrease in amount of FGFR biologicalactivity in the first sample compared to the reference sample indicatesthat the candidate substance is capable of inhibiting KLβ.

In another aspect, the invention provides methods of determining whethera candidate substance promotes KLβ biological activity, said methodcomprising: (a) contacting a candidate substance with a first samplecomprising FGFR and KLβ, and (b) comparing amount of FGFR biologicalactivity in the sample with amount of FGFR biological activity in areference sample comprising similar amounts of KLβ and FGFR as the firstsample but that has not been contacted with said candidate substance,whereby an increase in amount of FGFR biological activity in the firstsample compared to the reference sample indicates that the candidatesubstance is capable of promoting KLβ binding to FGFR.

FGFR biological activities are described herein. In some embodiments,FGFR, FGF, KLβ are in an amount effective for FGFR biological activity

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A to 1E: KLβ forms a complex with FGF19, FGFR4, and heparin. FIG.1A FGF19 (0.5 μg), heparin (0.5 μg), and different FGFR-Fc fusionproteins (0.5 μg) were incubated in KLβΔTM-conditioned media for 18hours at 4° C. The protein interactions were determined by proteinA-agarose precipitation and immunoblot analyses. FIG. 1B KLβΔTM- orcontrol-conditioned medium was incubated in the presence or the absenceof FGF19 (0.5 μg), heparin (0.5 μg), or the FGFR4-Fc fusion protein (0.5μg) for 18 hours at 4° C. The protein interactions were determined byprotein A-agarose precipitation and immunoblot analyses. FIG. 1C andFIG. 1D Lysates from HEK293 cells transfected with empty (controlvector), FGFR4, KLβ-Flag, or a combination of FGFR4 and KLβ-Flagexpression vectors were incubated in the presence or absence of heparinand FGF19. The protein interactions were analyzed by FGFR4 FIG. 1C andKLβ-Flag FIG. 1D immunoprecipitation and immunoblotting. FIG. 1E TheFGFR4-KLβ interaction in HEPG2 cells lysates was analyzed byimmunoprecipitation and immunoblotting.

FIGS. 2A to 2D: KLβ is required for FGF19 signaling. The effect of KLβon FGF19 signaling was analyzed using HEK293 cells transfected withempty (control vector) FIG. 2A, KLβ FIG. 2B, FGFR4 FIG. 2C, or acombination of FGFR4 and KLβ expression vectors FIG. 2D. The transfectedcells were incubated with vehicle (PBS) or FGF19 (0-500 ng/mL) for 10minutes, lysed, and FRS2 and ERK1/2 phosphorylation were analyzed byimmunoblot.

FIGS. 3A to 3H: KLβ is required for FGF19 downstream modulation of geneexpression. FIG. 3A FGF19 represses KLβ expression. Cell lines wereincubated with FGF19 (100 ng/mL; 0-24 hours) and KLβ expression levelswere analyzed by RT-PCR. All values were compared with KLβ expressionlevels in HEP3B cells at time 0. A triplicate set of data was analyzedfor each condition. Data are presented as the mean±SEM. (B-D) FGF19promotes expression of c-Fos, JunB, and c-Jun. Cell lines were incubatedwith FGF19 (100 ng/mL; 0-24 hours) and c-Fos FIG. 3B, JunB FIG. 3C, andc-Jun FIG. 3D expression were analyzed by RT-PCR. The values representthe relative fold increase in the expression of a particular genecompared with its expression before exposure to FGF19. FIG. 3E KLβ siRNAtransfection represses KLβ synthesis. HEP3B cells transfected with eachof four different KLβ siRNAs were analyzed for KLβ expression byimmunoblot. FIG. 3F Inhibition of KLβ expression by KLβ siRNAtransfection inhibits FGF19 signaling. HEP3B cells transfected with eachof four different KLB siRNAs were incubated with vehicle (PBS) or FGF19(100 ng/mL) for 10 minutes and analyzed for FRS2 and ERK1/2phosphorylation by immunoblot. FIG. 3G Inhibition of KLβ expression byKLβ siRNA transfection inhibits FGF19-mediated c-Fos induction. HEP3Bcells transfected with each of four different KLβ siRNAs were incubatedwith FGF19 (100 ng/mL) for 90 minutes and KLβ and c-Fos expressionlevels were analyzed by RT-PCR. The values represent the relativeexpression of each particular gene compared with that of cellstransfected with control siRNA. FIG. 3H HEK293 cells transfected witheither empty (control vector), KLβ, FGFR4, or a combination of FGFR4 andKLβ expression vectors were incubated with PBS or FGF19 (100 ng/mL) for90 minutes; c-Fos expression was analyzed by RT-PCR. The valuesrepresent the fold increase in c-Fos expression compared with theexpression levels before cells were exposed to FGF19.

FIGS. 4A to 4F: KLβ and FGFR4 distribution determine FGF19tissue-specific activity. KLβ and FGFR4 distribution in human tissues.Whisker-box plots showing KLβ FIG. 4A and FGFR4 FIG. 4B expression inhuman tissues, as determined by mRNA analysis of the BioExpressdatabase. The center line indicates the median; the box represents theinter-quartile range between the first and third quartiles. Whiskersextend from the inter-quartile to the positions of extreme values. KLβFIG. 4C and FGFR4 FIG. 4D expression in a panel of mouse tissues weredetermined by RT-PCR. The value for each organ represents the meanexpression (n=3 mice), fold relative to the expression level observed inbrain tissues. FIG. 4E The tissue specificity of FGF19 in vivo wasdetermined by analyzing c-Fos expression in various organ tissues 30minutes after mice (n=3) were injected with PBS or FGF19 (1 mg/kg). Thevalues represent c-Fos expression in mice injected with FGF19, comparedwith the expression levels in mice injected with PBS. FIG. 4F CYP7A1expression in mouse livers 30 minutes after injection with FGF19 (1mg/kg) or PBS. The values represent the CYP7A1 expression in miceinjected with FGF19 compared with the expression found in mice injectedwith PBS. A triplicate set of data was analyzed for each condition. Dataare presented as the mean±SEM.

FIGS. 5A to 5C: KLβ is required for FGF19 downstream modulation of geneexpression in HEPG2 cells. FIG. 5A KLB siRNA transfection represses KLβsynthesis. Expression of KLβ in HEPG2 cells transfected with each offour different KLβ siRNAs was analyzed by immunoblot. FIG. 5B KLβ siRNAtransfection inhibits FGF19 signaling. HEPG2 cells transfected with eachof four different KLβ siRNAs were incubated with PBS or FGF19 (100ng/mL) for 10 minutes, lysed, and analyzed for FRS2 phosphorylationlevels by immunoblot. FIG. 5C KLβ siRNA transfection inhibits FGF19mediated c-Fos induction. HEP3B cells transfected with each of fourdifferent KLβ siRNAs were incubated with FGF19 (100 ng/mL) for 90minutes and KLβ and c-Fos expression were analyzed by RT-PCR. The valuesrepresent the expression of each gene compared with its expression incontrol siRNA-transfected cells.

FIG. 6: Treatment with anti-KLβ antibody inhibits FGF19-mediated c-Fosinduction. HEPG2 cells were treated with a control antibody or apolyclonal anti-KLβ antibody that was raised against mouse KLβ butcross-reacts with human KLβ (10 μg/ml). FGF19 stimulated c-Fos inductionwas measured by RT-PCR. The anti-KLβ antibody treatment inhibited theFGF19-mediated c-Fos induction whereas the control antibody did not haveany significant effect.

FIG. 7: KLβ active site mutation inhibits FGF19 pathway activation.HEK293 cells were untransfected or transfected with the KLβ E416A(active site) or the KLβ E693A (non-active site) mutant.FGF19-stimulated activity was assessed by phosphorylated FRS2 andphosphorylated ERK1/2 immunodetection. FGF19 treatment (100 ng/ml; 10min) yielded an increased in phosphorylated FRS2 and phosphorylatedERK1/2 signal in wildtype (wt) KLβ transfected cells whereas thesesignals were undetectable in untransfected cells. The FRS2 and ERK1/2phosphorylation in KLβ E693A mutant transfected cells was comparable tothe FRS2 and ERK1/2 phosphorylation in the wildtype KLβ-transfectedcells. The FGF19 stimulation in KLβ E416A transfected cells was greatlyreduced compared to the wildtype KLβ. These findings corroborate theenhancement of FGF19 signaling by KLβ and further suggest therequirement of KLβ enzymatic activity for FGF19 signaling.

FIG. 8: KLβ antibody treatment inhibits FGF19-dependent c-Fos inductionin mouse liver. FGF19-dependent c-Fos induction was measured in theliver of a mouse treated with a KLβ antibody (2.2 mg/kg) for 0, 3, 9 or24 hours. Anti-KLβ antibody treatment for 3, 9 or 24 hours before aFGF19 injection (1 mg/kg) reduced the liver specific FGF19-mediatedc-Fos induction by 58%, 68% and 91% respectively.

FIG. 9: Expression of Ku mRNA was determined in tumor tissues.

FIG. 10: Expression of FGFR4 mRNA was determined in tumor tissues.

FIG. 11: depicts an exemplary KLβ nucleic acid sequence (SEQ ID NO:1).

FIG. 12: depicts an exemplary KLβ amino acid sequence (SEQ ID NO:2).

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the invention provides compositions and methods based onbinding KLβ. KLβ binding agents, as described herein, provide importanttherapeutic and diagnostic agents for use in targeting pathologicalconditions associated with expression and/or activity of theKLβ-FGF19-FGFR4 pathways. Accordingly, the invention provides methods,compositions, kits and articles of manufacture related to KLβ binding.In another aspect, the invention provides methods based on detection ofKLβ of FGF (such as FGF19) and/or FGFR (such as FGFR4).

General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Sambrook et al., MolecularCloning: A Laboratory Manual 3rd. edition (2001) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. CURRENT PROTOCOLS INMOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (2003)); the seriesMETHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICALAPPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)),Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMALCELL CULTURE (R. I. Freshney, ed. (1987)).

DEFINITIONS

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe antibody nucleic acid. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the antibody where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat”, and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain may include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g. residues 82a, 82b, and 82c, etc according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues may be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence.

The phrase “substantially similar,” or “substantially the same”, as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (generally one associated with an antibody and the otherassociated with a reference/comparator antibody) such that one of skillin the art would consider the difference between the two values to be oflittle or no biological and/or statistical significance within thecontext of the biological characteristic measured by said values (e.g.,Kd values). The difference between said two values is preferably lessthan about 50%, preferably less than about 40%, preferably less thanabout 30%, preferably less than about 20%, preferably less than about10% as a function of the value for the reference/comparator antibody.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative embodiments are describedin the following.

In one embodiment, the “Kd” or “Kd value” according to this invention ismeasured by a radiolabeled antigen binding assay (RIA) performed withthe Fab version of an antibody of interest and its antigen as describedby the following assay that measures solution binding affinity of Fabsfor antigen by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (Chen, et al., (1999) J. Mol Biol 293:865-881). Toestablish conditions for the assay, microtiter plates (Dynex) are coatedovernight with 5 ug/ml of a capturing anti-Fab antibody (Cappel Labs) in50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v)bovine serum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbant plate (Nunc #269620), 100 pMor 26 pM [¹²⁵I]-antigen antigen are mixed with serial dilutions of a Fabof interest (e.g., consistent with assessment of an anti-VEGF antibody,Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599). The Fab ofinterest is then incubated overnight; however, the incubation maycontinue for a longer period (e.g., 65 hours) to insure that equilibriumis reached. Thereafter, the mixtures are transferred to the captureplate for incubation at room temperature (e.g., for one hour). Thesolution is then removed and the plate washed eight times with 0.1%Tween-20 in PBS. When the plates have dried, 150 ul/well of scintillant(MicroScint-20; Packard) is added, and the plates are counted on aTopcount gamma counter (Packard) for ten minutes. Concentrations of eachFab that give less than or equal to 20% of maximal binding are chosenfor use in competitive binding assays. According to another embodimentthe Kd or Kd value is measured by using surface plasmon resonance assaysusing a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway,N.J.) at 25 C with immobilized antigen CM5 chips at ˜10 response units(RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcoreInc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, into 5 ug/ml (˜0.2 uM) before injection at a flow rate of5u1/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1M ethanolamine is injectedto block unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%Tween 20 (PBST) at 25° C. at a flow rate of approximately 25 ul/min.Association rates (k_(on)) and dissociation rates (k_(off)) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneous fitting the associationand dissociation sensorgram. The equilibrium dissociation constant (Kd)is calculated as the ratio k_(off)/k_(on). See, e.g., Chen, Y., et al.,(1999) J. Mol Biol 293:865-881. If the on-rate exceeds 10⁶ M⁻¹ S⁻¹ bythe surface plasmon resonance assay above, then the on-rate can bedetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence emission intensity (excitation=295nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-Aminco spectrophotometer (ThermoSpectronic) with a stir red cuvette.

An “on-rate” or “rate of association” or “association rate” or “k_(on)”according to this invention can also be determined with the same surfaceplasmon resonance technique described above using a BIAcore™-2000 or aBIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25 C with immobilizedantigen CM5 chips at ˜10 response units (RU). Briefly, carboxymethylateddextran biosensor chips (CM5, BIAcore Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, into 5 ug/ml (˜0.2uM) before injection at a flow rate of 5 ul/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at25° C. at a flow rate of approximately 25 ul/min. Association rates(k_(on)) and dissociation rates (k_(off)) are calculated using a simpleone-to-one Langmuir binding model (BIAcore Evaluation Software version3.2) by simultaneous fitting the association and dissociationsensorgram. The equilibrium dissociation constant (Kd) was calculated asthe ratio k_(off)/k_(on). See, e.g., Chen, Y., et al., (1999) J. MolBiol 293:865-881. However, if the on-rate exceeds 10⁶ M⁻¹ S⁻¹ by thesurface plasmon resonance assay above, then the on-rate is preferablydetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence emission intensity (excitation=295nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred cuvette.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “recombinantvectors”). In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase, or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter synthesis, such as by conjugation with a label. Other types ofmodifications include, for example, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotide(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid or semi-solidsupports. The 5′ and 3′ terminal OH can be phosphorylated or substitutedwith amines or organic capping group moieties of from 1 to 20 carbonatoms. Other hydroxyls may also be derivatized to standard protectinggroups. Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars suchas arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs and a basic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), “(O)NR₂ (“amidate”), P(O)R,P(O)OR′, CO or CH₂ (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

“Oligonucleotide,” as used herein, generally refers to short, generallysingle stranded, generally synthetic polynucleotides that are generally,but not necessarily, less than about 200 nucleotides in length. Theterms “oligonucleotide” and “polynucleotide” are not mutually exclusive.The description above for polynucleotides is equally and fullyapplicable to oligonucleotides.

The term “Klotho beta” (interchangeably termed “KLβ” or “Beta Klotho” orβKlotho”), as used herein, refers, unless specifically or contextuallyindicated otherwise, to any native or variant (whether native orsynthetic) KLβ polypeptide. The term “native sequence” specificallyencompasses naturally occurring truncated or secreted forms (e.g., anextracellular domain sequence), naturally occurring variant forms (e.g.,alternatively spliced forms) and naturally-occurring allelic variants.The term “wild type KLβ” generally refers to a polypeptide comprisingthe amino acid sequence of a naturally occurring KLβ protein. The term“wild type KLβ sequence” generally refers to an amino acid sequencefound in a naturally occurring KLβ.

The term “FGF19” (interchangeably termed “Fibroblast growth factor 19”),as used herein, refers, unless specifically or contextually indicatedotherwise, to any native or variant (whether native or synthetic) KLβpolypeptide. The term “native sequence” specifically encompassesnaturally occurring truncated or secreted forms (e.g., an extracellulardomain sequence), naturally occurring variant forms (e.g., alternativelyspliced forms) and naturally-occurring allelic variants. The term “wildtype KLβ” generally refers to a polypeptide comprising the amino acidsequence of a naturally occurring KLβ protein. The term “wild type KLβsequence” generally refers to an amino acid sequence found in anaturally occurring KLβ.

A “FGF19 antagonist” refers to a molecule capable of neutralizing,blocking, inhibiting, abrogating, reducing or interfering with theactivities of a FGF19 including, for example, binding KLβ (optionally inconjunction with heparin), binding FGFR4 (optionally in conjunction withheparin), binding KLβ and FGFR4 (optionally in conjunction withheparin), promoting FGF19-mediated induction of cFos, Junb and/or Junc(in vitro or in vivo), promoting FGFR4 and/or FGF19 down streamsignaling (including but not limited to FRS2 phosphorylation, ERK1/2phosphorylation and Wnt pathway activation), and/or promotion of anybiologically relevant FGF19 and/or FGFR4 biological pathway, and/orpromotion of a tumor, cell proliferative disorder or a cancer; and/orpromotion of a disorder associated with FGF19 expression and/or activity(such as increased FGF19 expression and/or activity). FGF19 antagonistsinclude antibodies and antigen-binding fragments thereof, proteins,peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides,oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics,pharmacological agents and their metabolites, transcriptional andtranslation control sequences, and the like. Antagonists also includesmall molecule inhibitors of a protein, and fusions proteins, receptormolecules and derivatives which bind specifically to protein therebysequestering its binding to its target, antagonist variants of theprotein, siRNA molecules directed to a protein, antisense moleculesdirected to a protein, RNA aptamers, and ribozymes against a protein. Insome embodiments, the FGF19 antagonist is a molecule which binds toFGF19 and neutralizes, blocks, inhibits, abrogates, reduces orinterferes with a biological activity of FGF19.

A “KLβ antagonist” refers to a molecule capable of neutralizing,blocking, inhibiting, abrogating, reducing or interfering with theactivities of a KLβ including, for example, binding FGFR (e.g., FGFR4)(optionally in conjunction with heparin), binding FGF (e.g. FGF19)(optionally in conjunction with heparin), binding FGFR4 and FGF19(optionally in conjunction with heparin), promoting FGF19-mediatedinduction of cFos, Junb and/or Junc (in vitro or in vivo), promotingFGFR4 and/or FGF19 down stream signaling (including but not limited toFRS2 phosphorylation, ERK1/2 phosphorylation and Wnt pathwayactivation), and/or promotion of any biologically relevant KLβ and/orFGFR4 biological pathway, and/or promotion of a tumor, cellproliferative disorder or a cancer; and/or promotion of a disorderassociated with KLβ expression and/or activity (such as increased KLβexpression and/or activity). KLβ antagonists include antibodies andantigen-binding fragments thereof, proteins, peptides, glycoproteins,glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleicacids, bioorganic molecules, peptidomimetics, pharmacological agents andtheir metabolites, transcriptional and translation control sequences,and the like. Antagonists also include small molecule inhibitors of aprotein, and fusions proteins, receptor molecules and derivatives whichbind specifically to protein thereby sequestering its binding to itstarget, antagonist variants of the protein, siRNA molecules directed toa protein, antisense molecules directed to a protein, RNA aptamers, andribozymes against a protein. In some embodiments, the KLβ antagonist isa molecule which binds to KLβ and neutralizes, blocks, inhibits,abrogates, reduces or interferes with a biological activity of KLβ.

The term “FGFR4” (interchangeably termed “Fibroblast growth factorreceptor 4”), as used herein, refers, unless specifically orcontextually indicated otherwise, to any native or variant (whethernative or synthetic) FGFR4 polypeptide. The term “native sequence”specifically encompasses naturally occurring truncated or secreted forms(e.g., an extracellular domain sequence), naturally occurring variantforms (e.g., alternatively spliced forms) and naturally-occurringallelic variants. The term “wild type FGFR4” generally refers to apolypeptide comprising the amino acid sequence of a naturally occurringFGFR4 protein. The term “wild type FGFR4 sequence” generally refers toan amino acid sequence found in a naturally occurring FGFR4.

A “FGFR antagonist” refers to a molecule capable of neutralizing,blocking, inhibiting, abrogating, reducing or interfering with theactivities of a FGF receptor (“FGFR”) including, for example, bindingKLβ (optionally in conjunction with heparin), binding FGF (e.g., FGF19)(optionally in conjunction with heparin), binding KLβ and FGF (e.g.,FGF19) (optionally in conjunction with heparin), promotingFGF19-mediated induction of cFos, Junb and/or Junc (in vitro or invivo), promoting FGFR and/or FGF down stream signaling (including butnot limited to FRS2 phosphorylation, ERK1/2 phosphorylation and Wntpathway activation), and/or promotion of any biologically relevant FGFand/or FGFR biological pathway, and/or promotion of a tumor, cellproliferative disorder or a cancer; and/or promotion of a disorderassociated with FGFR expression and/or activity (such as increased FGFRexpression and/or activity). FGFR antagonists include antibodies andantigen-binding fragments thereof, proteins, peptides, glycoproteins,glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleicacids, bioorganic molecules, peptidomimetics, pharmacological agents andtheir metabolites, transcriptional and translation control sequences,and the like. Antagonists also include small molecule inhibitors of aprotein, and fusions proteins, receptor molecules and derivatives whichbind specifically to protein thereby sequestering its binding to itstarget, antagonist variants of the protein, siRNA molecules directed toa protein, antisense molecules directed to a protein, RNA aptamers, andribozymes against a protein. In some embodiments, the FGFR antagonist(e.g., FGFR4 antagonist) is a molecule which binds to FGFR andneutralizes, blocks, inhibits, abrogates, reduces or interferes with abiological activity of FGFR.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (for e.g., fulllength or intact monoclonal antibodies), polyclonal antibodies,multivalent antibodies, multispecific antibodies (e.g., bispecificantibodies so long as they exhibit the desired biological activity) andmay also include certain antibody fragments (as described in greaterdetail herein). An antibody can be human, humanized and/or affinitymatured.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, Fifth Edition, National Institute ofHealth, Bethesda, Md. (1991)). The constant domains are not involveddirectly in binding an antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. In a two-chain Fv species, thisregion consists of a dimer of one heavy- and one light-chain variabledomain in tight, non-covalent association. In a single-chain Fv species,one heavy- and one light-chain variable domain can be covalently linkedby a flexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. Theheavy-chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion preferably retains at least one, preferably most orall, of the functions normally associated with that portion when presentin an intact antibody. Examples of antibody fragments include Fab, Fab′,F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chainantibody molecules; and multispecific antibodies formed from antibodyfragments. In one embodiment, an antibody fragment comprises an antigenbinding site of the intact antibody and thus retains the ability to bindantigen. In another embodiment, an antibody fragment, for example onethat comprises the Fc region, retains at least one of the biologicalfunctions normally associated with the Fc region when present in anintact antibody, such as FcRn binding, antibody half life modulation,ADCC function and complement binding. In one embodiment, an antibodyfragment is a monovalent antibody that has an in vivo half lifesubstantially similar to an intact antibody. For e.g., such an antibodyfragment may comprise on antigen binding arm linked to an Fc sequencecapable of conferring in vivo stability to the fragment.

The term “hypervariable region”, “HVR”, or “HV”, when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six hypervariable regions; three in the VH (H1, H2, H3), andthree in the VL (L1, L2, L3). A number of hypervariable regiondelineations are in use and are encompassed herein. The KabatComplementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The AbM hypervariable regions representa compromise between the Kabat CDRs and Chothia structural loops, andare used by Oxford Molecular's AbM antibody modeling software. The“contact” hypervariable regions are based on an analysis of theavailable complex crystal structures.

Hypervariable regions may comprise “extended hypervariable regions” asfollows: 24-36 (L1), 46-56 (L2) and 89-97 (L3) in the VL and 26-35 (H1),49-65 or 50 to 65 (H2) and 93-102 (H3) in the VH. The variable domainresidues are numbered according to Kabat et al., supra for each of thesedefinitions.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994).

“Chimeric” antibodies (immunoglobulins) have a portion of the heavyand/or light chain identical with or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).Humanized antibody as used herein is a subset of chimeric antibodies.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFvsee Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

An “antigen” is a predetermined antigen to which an antibody canselectively bind. The target may be polypeptide, carbohydrate, nucleicacid, lipid, hapten or other naturally occurring or synthetic compound.Preferably, the target is a polypeptide.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues.

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). Preferred affinity matured antibodieswill have nanomolar or even picomolar affinities for the target.Affinity matured antibodies are produced by procedures known in the art.Marks et al. Bio/Technology 10:779-783 (1992) describes affinitymaturation by VH and VL domain shuffling. Random mutagenesis of CDRand/or framework residues is described by: Barbas et al. Proc Nat. Acad.Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995);Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J.Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.226:889-896 (1992).

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g. Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cellsis summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.Immunol 9:457-92 (1991). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, such as that described in U.S. Pat.No. 5,500,362 or 5,821,337 or Presta U.S. Pat. No. 6,737,056 may beperformed. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source, e.g. from blood.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII, and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domainInhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see review M. inDaëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. The term also includesthe neonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)) and regulates homeostasis ofimmunoglobulins.

WO00/42072 (Presta) describes antibody variants with improved ordiminished binding to FcRs. The content of that patent publication isspecifically incorporated herein by reference. See, also, Shields et al.J. Biol. Chem. 9(2): 6591-6604 (2001).

Methods of measuring binding to FcRn are known (see, e.g., Ghetie 1997,Hinton 2004). Binding to human FcRn in vivo and serum half life of humanFcRn high affinity binding polypeptides can be assayed, e.g., intransgenic mice or transfected human cell lines expressing human FcRn,or in primates administered with the Fc variant polypeptides.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996), may be performed.

Polypeptide variants with altered Fc region amino acid sequences andincreased or decreased C1q binding capability are described in U.S. Pat.No. 6,194,551B1 and WO99/51642. The contents of those patentpublications are specifically incorporated herein by reference. See,also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it binds. Preferredblocking antibodies or antagonist antibodies substantially or completelyinhibit the biological activity of the antigen.

A “biological sample” (interchangeably termed “sample” or “tissue orcell sample”) encompasses a variety of sample types obtained from anindividual and can be used in a diagnostic or monitoring assay. Thedefinition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom, and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides, or embedding in a semi-solid or solid matrix forsectioning purposes. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples. The sourceof the biological sample may be solid tissue as from a fresh, frozenand/or preserved organ or tissue sample or biopsy or aspirate; blood orany blood constituents; bodily fluids such as cerebral spinal fluid,amniotic fluid, peritoneal fluid, or interstitial fluid; cells from anytime in gestation or development of the subject. In some embodiments,the biological sample is obtained from a primary or metastatic tumor.The biological sample may contain compounds which are not naturallyintermixed with the tissue in nature such as preservatives,anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.

For the purposes herein a “section” of a tissue sample is meant a singlepart or piece of a tissue sample, e.g. a thin slice of tissue or cellscut from a tissue sample. It is understood that multiple sections oftissue samples may be taken and subjected to analysis according to thepresent invention. In some embodiments, the same section of tissuesample is analyzed at both morphological and molecular levels, or isanalyzed with respect to both protein and nucleic acid.

The word “label” when used herein refers to a compound or compositionwhich is conjugated or fused directly or indirectly to a reagent such asa nucleic acid probe or an antibody and facilitates detection of thereagent to which it is conjugated or fused. The label may itself bedetectable (e.g., radioisotope labels or fluorescent labels) or, in thecase of an enzymatic label, may catalyze chemical alteration of asubstrate compound or composition which is detectable.

A “medicament” is an active drug to treat the disorder in question orits symptoms, or side effects.

A “disorder” or “disease” is any condition that would benefit fromtreatment with a substance/molecule or method of the invention. Thisincludes chronic and acute disorders or diseases including thosepathological conditions which predispose the mammal to the disorder inquestion. Non-limiting examples of disorders to be treated hereininclude malignant and benign tumors; carcinoma, blastoma, and sarcoma.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer”, “cancerous”, “cellproliferative disorder”, “proliferative disorder” and “tumor” are notmutually exclusive as referred to herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include butare not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia. More particular examples of such cancers include squamous cellcancer, small-cell lung cancer, pituitary cancer, esophageal cancer,astrocytoma, soft tissue sarcoma, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma, brain cancer, endometrial cancer,testis cancer, cholangiocarcinoma, gallbladder carcinoma, gastriccancer, melanoma, and various types of head and neck cancer.Dysregulation of angiogenesis can lead to many disorders that can betreated by compositions and methods of the invention. These disordersinclude both non-neoplastic and neoplastic conditions. Neoplasticsinclude but are not limited those described above. Non-neoplasticdisorders include but are not limited to undesired or aberranthypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis, psoriaticplaques, sarcoidosis, atherosclerosis, atherosclerotic plaques, diabeticand other proliferative retinopathies including retinopathy ofprematurity, retrolental fibroplasia, neovascular glaucoma, age-relatedmacular degeneration, diabetic macular edema, cornealneovascularization, corneal graft neovascularization, corneal graftrejection, retinal/choroidal neovascularization, neovascularization ofthe angle (rubeosis), ocular neovascular disease, vascular restenosis,arteriovenous malformations (AVM), meningioma, hemangioma, angiofibroma,thyroid hyperplasias (including Grave's disease), corneal and othertissue transplantation, chronic inflammation, lung inflammation, acutelung injury/ARDS, sepsis, primary pulmonary hypertension, malignantpulmonary effusions, cerebral edema (e.g., associated with acutestroke/closed head injury/trauma), synovial inflammation, pannusformation in RA, myositis ossificans, hypertropic bone formation,osteoarthritis (OA), refractory ascites, polycystic ovarian disease,endometriosis, 3rd spacing of fluid diseases (pancreatitis, compartmentsyndrome, burns, bowel disease), uterine fibroids, premature labor,chronic inflammation such as IBD (Crohn's disease and ulcerativecolitis), renal allograft rejection, inflammatory bowel disease,nephrotic syndrome, undesired or aberrant tissue mass growth(non-cancer), hemophilic joints, hypertrophic scars, inhibition of hairgrowth, Osler-Weber syndrome, pyogenic granuloma retrolentalfibroplasias, scleroderma, trachoma, vascular adhesions, synovitis,dermatitis, preeclampsia, ascites, pericardial effusion (such as thatassociated with pericarditis), and pleural effusion.

The term “wasting” disorders (e.g., wasting syndrome, cachexia,sarcopenia) refers to a disorder caused by undesirable and/or unhealthyloss of weight or loss of body cell mass. In the elderly as well as inAIDS and cancer patients, wasting disease can result in undesired lossof body weight, including both the fat and the fat-free compartments.Wasting diseases can be the result of inadequate intake of food and/ormetabolic changes related to illness and/or the aging process. Cancerpatients and AIDS patients, as well as patients following extensivesurgery or having chronic infections, immunologic diseases,hyperthyroidism, Crohn's disease, psychogenic disease, chronic heartfailure or other severe trauma, frequently suffer from wasting diseasewhich is sometimes also referred to as cachexia, a metabolic and,sometimes, an eating disorder. Cachexia is additionally characterized byhypermetabolism and hypercatabolism. Although cachexia and wastingdisease are frequently used interchangeably to refer to wastingconditions, there is at least one body of research which differentiatescachexia from wasting syndrome as a loss of fat-free mass, andparticularly, body cell mass (Mayer, 1999, J. Nutr. 129(1SSuppl.):256S-259S). Sarcopenia, yet another such disorder which canaffect the aging individual, is typically characterized by loss ofmuscle mass. End stage wasting disease as described above can develop inindividuals suffering from either cachexia or sarcopenia.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects of treatment includepreventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis. Insome embodiments, antibodies are used to delay development of a diseaseor disorder.

An “anti-angiogenesis agent” or “angiogenesis inhibitor” refers to asmall molecular weight substance, a polynucleotide, a polypeptide, anisolated protein, a recombinant protein, an antibody, or conjugates orfusion proteins thereof, that inhibits angiogenesis, vasculogenesis, orundesirable vascular permeability, either directly or indirectly. Forexample, an anti-angiogenesis agent is an antibody or other antagonistto an angiogenic agent as defined above, e.g., antibodies to VEGF,antibodies to VEGF receptors, small molecules that block VEGF receptorsignaling (e.g., PTK787/ZK2284, SU6668, SUTENT/SU11248 (sunitinibmalate), AMG706). Anti-angiogensis agents also include nativeangiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See, e.g.,Klagsbrun and D'Amore, Annu Rev. Physiol., 53:217-39 (1991); Streit andDetmar, Oncogene, 22:3172-3179 (2003) (e.g., Table 3 listinganti-angiogenic therapy in malignant melanoma); Ferrara & Alitalo,Nature Medicine 5(12):1359-1364 (1999); Tonini et al., Oncogene,22:6549-6556 (2003) (e.g., Table 2 listing antiangiogenic factors); and,Sato Int. J. Clin. Oncol., 8:200-206 (2003) (e.g., Table 1 listsAnti-angiogenic agents used in clinical trials).

An “individual” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, farm animals (such ascows), sport animals, pets (such as cats, dogs and horses), primates,mice and rats.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

A “therapeutically effective amount” of a substance/molecule of theinvention, agonist or antagonist may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the substance/molecule, agonist or antagonist to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of thesubstance/molecule, agonist or antagonist are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typicallybut not necessarily, since a prophylactic dose is used in subjects priorto or at an earlier stage of disease, the prophylactically effectiveamount will be less than the therapeutically effective amount.

“Conditions related to obesity” refer to conditions which are the resultof or which are exasperated by obesity, such as, but not limited todermatological disorders such as infections, varicose veins, Acanthosisnigricans, and eczema, exercise intolerance, type II diabetes mellitus,insulin resistance, hypertension, hypercholesterolemia, cholelithiasis,osteoarthritis, orthopedic injury, thromboembolic disease, cancer, andcoronary (or cardiovascular) heart disease, particular thosecardiovascular conditions associated with high triglycerides and freefatty acids in an individual.

“Obesity” refers to a condition whereby a mammal has a Body Mass Index(BMI), which is calculated as weight (kg) per height² (meters), of atleast 25.9. Conventionally, those persons with normal weight have a BMIof 19.9 to less than 25.9. The obesity herein may be due to any cause,whether genetic or environmental. Examples of disorders that may resultin obesity or be the cause of obesity include overeating and bulimia,polycystic ovarian disease, craniopharyngioma, the Prader-WilliSyndrome, Frohlich's syndrome, Type II diabetes, GH-deficient subjects,normal variant short stature, Turner's syndrome, and other pathologicalconditions showing reduced metabolic activity or a decrease in restingenergy expenditure as a percentage of total fat-free mass, e.g.,children with acute lymphoblastic leukemia.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin,vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof, and the variousantitumor or anticancer agents disclosed below. Other cytotoxic agentsare described below. A tumoricidal agent causes destruction of tumorcells.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTIN®), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g.,Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,ADRIAMYCINO doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine(VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine(NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromefthylornithine(DMF®); retinoids such as retinoic acid; capecitabine (XELODA®);pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Also included in this definition are anti-hormonal agents that act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer, and are often in the form of systemic, orwhole-body treatment. They may be hormones themselves. Examples includeanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and FARESTON® toremifene;anti-progesterones; estrogen receptor down-regulators (ERDs); agentsthat function to suppress or shut down the ovaries, for example,leutinizing hormone-releasing hormone (LHRH) agonists such as LUPRON®and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetateand tripterelin; other anti-androgens such as flutamide, nilutamide andbicalutamide; and aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. Inaddition, such definition of chemotherapeutic agents includesbisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid/zoledronate,FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, orACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolanenucleoside cytosine analog); antisense oligonucleotides, particularlythose that inhibit expression of genes in signaling pathways implicatedin abherant cell proliferation, such as, for example, PKC-alpha, Raf,H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such asTHERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN®topoisomerase 1 inhibitor; ABARELIX® rmRH; lapatinib ditosylate (anErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also knownas GW572016); and pharmaceutically acceptable salts, acids orderivatives of any of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell (such as a cell expressingKLβ) either in vitro or in vivo. Thus, the growth inhibitory agent maybe one which significantly reduces the percentage of cells (such as acell expressing KLβ) in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce G1 arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), taxanes, and topoisomerase II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders:Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel anddocetaxel) are anticancer drugs both derived from the yew tree.Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the Europeanyew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-MyersSquibb). Paclitaxel and docetaxel promote the assembly of microtubulesfrom tubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

“Doxorubicin” is an anthracycline antibiotic. The full chemical name ofdoxorubicin is(8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione.

The term “Fc region-comprising polypeptide” refers to a polypeptide,such as an antibody or immunoadhesin (see definitions below), whichcomprises an Fc region. The C-terminal lysine (residue 447 according tothe EU numbering system) of the Fc region may be removed, for example,during purification of the polypeptide or by recombinant engineering thenucleic acid encoding the polypeptide. Accordingly, a compositioncomprising a polypeptide having an Fc region according to this inventioncan comprise polypeptides with K447, with all K447 removed, or a mixtureof polypeptides with and without the K447 residue.

KLβ

KLβ is a transmembrane protein comprising an extracellular domaincontaining two regions with homology to those in family 1 glycosidases,a transmembrane domain, and a short intracellular hydrophilic tail atthe carboxy terminus. Human KLβ protein is a 1043 amino acid protein andcontains the following regions: signal peptide (amino acids 1-51);glycosidase (amino acids 77-508); glycosidase (amino acids 517-967);transmembrane (amino acids 996-1012); and cytoplasmic domain (aminoacids 1013-1043). KLβ nucleic acid and amino acid sequences are known inthe art and are further discussed herein. Nucleic acid sequence encodingthe KLβ can be designed using the amino acid sequence of the desiredregion of KLβ. Alternatively, the cDNA sequence (or fragments thereof)of KLβ can be used. The accession number of human KLβ is NM_(—)175737,and the accession number of mouse KLβ is NM_(—)031180. Additionalexemplary KLβ sequences are, e.g., shown in FIGS. 17 and 18, anddescribed, for example, in Ito et al. (2000) Mech Dev 98:115-119.

KLβ Modulators

Modulators of KLβ are molecules that modulate the activity of KLβ, e.g.,agonists and antagonists. The term “KLβ agonist” is defined in thecontext of the biological role of KLβ. In certain embodiments, agonistspossess the biological activities of a KLβ, as defined above. In someembodiments, KLβ agonists bind FGFR4 (optionally in conjunction withheparin), bind FGF19 (optionally in conjunction with heparin), bindFGFR4 and FGF19 (optionally in conjunction with heparin), promoteFGF19-mediated induction of cFos, Junb and/or Junc (in vitro or invivo), promote FGFR4 and/or FGF19 down stream signaling (including butnot limited to FRS2 phosphorylation, ERK1/2 phosphorylation and Wntpathway activation), and/or promotion of any biologically relevant KLβand/or FGFR4 biological pathway.

KLβ modulators are known in the art, and some are described andexemplified herein. An exemplary and non-limiting list of KLβantagonists (such as an anti-KLβ antibody) contemplated is providedherein under “Definitions.”

The modulators useful in the present invention can be characterized fortheir physical/chemical properties and biological functions by variousassays known in the art. In some embodiments, KLβ antagonists arecharacterized for any one or more of: binding to KLβ, reduction orblocking of FGFR4 activation, reduction or blocking of FGFR4 receptordownstream molecular signaling, inhibition of KLβ enzymatic activity(such as KLβ glycosidase activity), disruption or blocking of binding toFGF19, reduction and/or blocking of FGF19 downstream molecularsignaling, and/or treatment and/or prevention of a tumor, cellproliferative disorder or a cancer (such as hepatocellular carcinoma);and/or treatment or prevention of a disorder associated with KLβexpression and/or activity. Methods for characterizing KLβ antagonistsand agonists are known in the art, and some are described andexemplified herein.

FGFR Modulators

Modulators of FGFR are molecules that modulate the activity of FGFR,e.g., agonists and antagonists. A “FGFR antagonist” refers to a moleculecapable of neutralizing, blocking, inhibiting, abrogating, reducing orinterfering with the activities of a FGF receptor (“FGFR”) including,for example, binding KLβ (optionally in conjunction with heparin),binding FGF (e.g., FGF19) (optionally in conjunction with heparin),binding KLβ and FGF (e.g., FGF19) (optionally in conjunction withheparin), promoting FGF19-mediated induction of cFos, Junb and/or Junc(in vitro or in vivo), promoting FGFR and/or FGF down stream signaling(including but not limited to FRS2 phosphorylation, ERK1/2phosphorylation and Wnt pathway activation), and/or promotion of anybiologically relevant FGF and/or FGFR biological pathway, and/orpromotion of a tumor, cell proliferative disorder or a cancer; and/orpromotion of a disorder associated with FGFR expression and/or activity(such as increased FGFR expression and/or activity). FGFR antagonistsinclude antibodies and antigen-binding fragments thereof, proteins,peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides,oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics,pharmacological agents and their metabolites, transcriptional andtranslation control sequences, and the like. Antagonists also includesmall molecule inhibitors of a protein, and fusions proteins, receptormolecules and derivatives which bind specifically to protein therebysequestering its binding to its target, antagonist variants of theprotein, siRNA molecules directed to a protein, antisense moleculesdirected to a protein, RNA aptamers, and ribozymes against a protein. Insome embodiments, the FGFR antagonist (e.g., FGFR4 antagonist) is amolecule which binds to FGFR and neutralizes, blocks, inhibits,abrogates, reduces or interferes with a biological activity of FGFR.

FGFR modulators are known in the art. For example, FGFR small moleculeinhibitors are described in Manetti, F. and Botta, M., Curr. Pharm.Des., 9, 567-581 (2003). An example of a FGFR4 small molecule inhibitoris PD173074 (Pfizer, Inc. Groton Conn.). An exemplary and non-limitinglist of FGFR antagonists (such as an anti-FGFR antibody) contemplated isprovided herein under “Definitions.” Methods for characterizing FGFRantagonists are known in the art, and some are described and exemplifiedherein.

FGF19 Antagonists

A “FGF19 antagonist” refers to a molecule capable of neutralizing,blocking, inhibiting, abrogating, reducing or interfering with theactivities of a FGF19 including, for example, binding KLβ (optionally inconjunction with heparin), binding FGFR4 (optionally in conjunction withheparin), binding KLβ and FGFR4 (optionally in conjunction withheparin), promoting FGF19-mediated induction of cFos, Junb and/or Junc(in vitro or in vivo), promoting FGFR4 and/or FGF19 down streamsignaling (including but not limited to FRS2 phosphorylation, ERK1/2phosphorylation and Wnt pathway activation), and/or promotion of anybiologically relevant FGF19 and/or FGFR4 biological pathway, and/orpromotion of a tumor, cell proliferative disorder or a cancer; and/orpromotion of a disorder associated with FGF19 expression and/or activity(such as increased FGF19 expression and/or activity). FGF19 antagonistsinclude antibodies and antigen-binding fragments thereof, proteins,peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides,oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics,pharmacological agents and their metabolites, transcriptional andtranslation control sequences, and the like. Antagonists also includesmall molecule inhibitors of a protein, and fusions proteins, receptormolecules and derivatives which bind specifically to protein therebysequestering its binding to its target, antagonist variants of theprotein, siRNA molecules directed to a protein, antisense moleculesdirected to a protein, RNA aptamers, and ribozymes against a protein. Insome embodiments, the FGF19 antagonist is a molecule which binds toFGF19 and neutralizes, blocks, inhibits, abrogates, reduces orinterferes with a biological activity of FGF19.

FGF19 antagonists are known in the art. An exemplary and non-limitinglist of FGF19 antagonists (such as an anti-FGFR antibody) contemplatedis provided herein under “Definitions.” Methods for characterizing FGFRantagonists are known in the art, and some are described and exemplifiedherein.

Antibodies

The antibodies are preferably monoclonal, although polyclonal antibodiesmay also be useful and are exemplified herein. Also encompassed withinthe scope of the invention are Fab, Fab′, Fab′-SH and F(ab′)₂ fragmentsof the antibodies provided herein. These antibody fragments can becreated by traditional means, such as enzymatic digestion, or may begenerated by recombinant techniques. Such antibody fragments may bechimeric or humanized. These fragments are useful for the diagnostic andtherapeutic purposes set forth below. Anti-KLβ antibodies are known inthe art, e.g., antibodies disclosed in Ito et al (2005) J Clin Invest115(8): 2202-2208; R&D Systems Catalog No. MAB3738. Anti-FGF19antibodies are disclosed in, e.g., WO2007/13693. The anti-FGF19 antibodymay be an antibody comprising (a) a light chain comprising (i) HVR-L1comprising the sequence KASQDINSFLA (SEQ ID NO:53); (ii) HVR-L2comprising the sequence RANRLVS (SEQ ID NO:54); and (iii) HVR-L3comprising the sequence LQYDEFPLT (SEQ ID NO:55), and (b) a heavy chaincomprising (i) HVR-H1 comprising the sequence GFSLTTYGVH (SEQ ID NO:56);(ii) HVR-H2 comprising the sequence GVIWPGGGTDYNAAFIS (SEQ ID NO:57);and (iii) HVR-H3 comprising the sequence VRKEYANLYA (SEQ ID NO:58).

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

The monoclonal antibodies can be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theprotein used for immunization. Antibodies to a given target generallyare raised in animals by multiple subcutaneous (sc) or intraperitoneal(ip) injections of target immunogen and an adjuvant. Target polypeptidemay be prepared using methods well-known in the art, some of which arefurther described herein. For example, recombinant production of proteinis described below. In one embodiment, animals are immunized with aderivative of antigen that contains the extracellular domain (ECD) ofthe target fused to the Fc portion of an immunoglobulin heavy chain. Inone embodiment, animals are immunized with a target polypeptide-IgG1fusion protein. Animals ordinarily are immunized against immunogenicconjugates or derivatives of target polypeptide with monophosphoryllipid A (MPL)/trehalose dicrynomycolate (TDM) (Ribi Immunochem.Research, Inc., Hamilton, Mont.) and the solution is injectedintradermally at multiple sites. Two weeks later the animals areboosted. 7 to 14 days later animals are bled and the serum is assayedfor anti-antigen titer. Animals are boosted until titer plateaus.

Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoadsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

The antibodies can be made by using combinatorial libraries to screenfor synthetic antibody clones with the desired activity or activities.In principle, synthetic antibody clones are selected by screening phagelibraries containing phage that display various fragments of antibodyvariable region (Fv) fused to phage coat protein. Such phage librariesare panned by affinity chromatography against the desired antigen.Clones expressing Fv fragments capable of binding to the desired antigenare adsorbed to the antigen and thus separated from the nonbindingclones in the library. The binding clones are then eluted from theantigen, and can be further enriched by additional cycles of antigenadsorption/elution. Any of the desired antibodies can be obtained bydesigning a suitable antigen screening procedure to select for the phageclone of interest followed by construction of a full length antibodyclone using the Fv sequences from the phage clone of interest andsuitable constant region (Fc) sequences described in Kabat et al.,Sequences of Proteins of Immunological Interest, Fifth Edition, NIHPublication 91-3242, Bethesda Md. (1991), vols. 1-3.

The antigen-binding domain of an antibody is formed from two variable(V) regions of about 110 amino acids, one each from the light (VL) andheavy (VH) chains, that both present three hypervariable loops orcomplementarity-determining regions (CDRs). Variable domains can bedisplayed functionally on phage, either as single-chain Fv (scFv)fragments, in which VH and VL are covalently linked through a short,flexible peptide, or as Fab fragments, in which they are each fused to aconstant domain and interact non-covalently, as described in Winter etal., Ann. Rev. Immunol., 12: 433-455 (1994). As used herein, scFvencoding phage clones and Fab encoding phage clones are collectivelyreferred to as “Fv phage clones” or “Fv clones”.

Repertoires of VH and VL genes can be separately cloned by polymerasechain reaction (PCR) and recombined randomly in phage libraries, whichcan then be searched for antigen-binding clones as described in Winteret al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunizedsources provide high-affinity antibodies to the immunogen without therequirement of constructing hybridomas. Alternatively, the naiverepertoire can be cloned to provide a single source of human antibodiesto a wide range of non-self and also self antigens without anyimmunization as described by Griffiths et al., EMBO J, 12: 725-734(1993). Finally, naive libraries can also be made synthetically bycloning the unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

Filamentous phage is used to display antibody fragments by fusion to theminor coat protein pIII. The antibody fragments can be displayed assingle chain Fv fragments, in which VH and VL domains are connected onthe same polypeptide chain by a flexible polypeptide spacer, e.g. asdescribed by Marks et al., J. Mol. Biol., 222: 581-597 (1991), or as Fabfragments, in which one chain is fused to pIII and the other is secretedinto the bacterial host cell periplasm where assembly of a Fab-coatprotein structure which becomes displayed on the phage surface bydisplacing some of the wild type coat proteins, e.g. as described inHoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991).

In general, nucleic acids encoding antibody gene fragments are obtainedfrom immune cells harvested from humans or animals. If a library biasedin favor of anti-antigen clones is desired, the subject is immunizedwith antigen polypeptide to generate an antibody response, and spleencells and/or circulating B cells other peripheral blood lymphocytes(PBLs) are recovered for library construction. In a preferredembodiment, a human antibody gene fragment library biased in favor ofanti-human clones is obtained by generating an anti-huma antibodyresponse in transgenic mice carrying a functional human immunoglobulingene array (and lacking a functional endogenous antibody productionsystem) such that immunization gives rise to B cells producing humanantibodies against antigen. The generation of human antibody-producingtransgenic mice is described below.

Additional enrichment for anti-antigen reactive cell populations can beobtained by using a suitable screening procedure to isolate B cellsexpressing antigen-specific membrane bound antibody, e.g., by cellseparation with antigen affinity chromatography or adsorption of cellsto fluorochrome-labeled antigen protein followed by flow-activated cellsorting (FACS).

Alternatively, the use of spleen cells and/or B cells or other PBLs froman unimmunized donor provides a better representation of the possibleantibody repertoire, and also permits the construction of an antibodylibrary using any animal (human or non-human) species in which antigenis not antigenic. For libraries incorporating in vitro antibody geneconstruction, stem cells are harvested from the subject to providenucleic acids encoding unrearranged antibody gene segments. The immunecells of interest can be obtained from a variety of animal species, suchas human, mouse, rat, lagomorpha, luprine, canine, feline, porcine,bovine, equine, and avian species, etc.

Nucleic acid encoding antibody variable gene segments (including VH andVL segments) are recovered from the cells of interest and amplified. Inthe case of rearranged VH and VL gene libraries, the desired DNA can beobtained by isolating genomic DNA or mRNA from lymphocytes followed bypolymerase chain reaction (PCR) with primers matching the 5′ and 3′ endsof rearranged VH and VL genes as described in Orlandi et al., Proc.Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse Vgene repertoires for expression. The V genes can be amplified from cDNAand genomic DNA, with back primers at the 5′ end of the exon encodingthe mature V-domain and forward primers based within the J-segment asdescribed in Orlandi et al. (1989) and in Ward et al., Nature, 341:544-546 (1989). However, for amplifying from cDNA, back primers can alsobe based in the leader exon as described in Jones et al., Biotechnol.,9: 88-89 (1991), and forward primers within the constant region asdescribed in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732(1989). To maximize complementarity, degeneracy can be incorporated inthe primers as described in Orlandi et al. (1989) or Sastry et al.(1989). Preferably, the library diversity is maximized by using PCRprimers targeted to each V-gene family in order to amplify all availableVH and VL arrangements present in the immune cell nucleic acid sample,e.g. as described in the method of Marks et al., J. Mol. Biol., 222:581-597 (1991) or as described in the method of Orum et al., NucleicAcids Res., 21: 4491-4498 (1993). For cloning of the amplified DNA intoexpression vectors, rare restriction sites can be introduced within thePCR primer as a tag at one end as described in Orlandi et al. (1989), orby further PCR amplification with a tagged primer as described inClackson et al., Nature, 352: 624-628 (1991).

Repertoires of synthetically rearranged V genes can be derived in vitrofrom V gene segments. Most of the human VH-gene segments have beencloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet.,3: 88-94 (1993); these cloned segments (including all the majorconformations of the H1 and H2 loop) can be used to generate diverse VHgene repertoires with PCR primers encoding H3 loops of diverse sequenceand length as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be made with all the sequencediversity focused in a long H3 loop of a single length as described inBarbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). HumanVκ and Vλ segments have been cloned and sequenced (reported in Williamsand Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used tomake synthetic light chain repertoires. Synthetic V gene repertoires,based on a range of VH and VL folds, and L3 and H3 lengths, will encodeantibodies of considerable structural diversity. Following amplificationof V-gene encoding DNAs, germline V-gene segments can be rearranged invitro according to the methods of Hoogenboom and Winter, J. Mol. Biol.,227: 381-388 (1992).

Repertoires of antibody fragments can be constructed by combining VH andVL gene repertoires together in several ways. Each repertoire can becreated in different vectors, and the vectors recombined in vitro, e.g.,as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo bycombinatorial infection, e.g., the loxP system described in Waterhouseet al., Nucl. Acids Res., 21: 2265-2266 (1993). The in vivorecombination approach exploits the two-chain nature of Fab fragments toovercome the limit on library size imposed by E. coli transformationefficiency. Naive VH and VL repertoires are cloned separately, one intoa phagemid and the other into a phage vector. The two libraries are thencombined by phage infection of phagemid-containing bacteria so that eachcell contains a different combination and the library size is limitedonly by the number of cells present (about 10¹² clones). Both vectorscontain in vivo recombination signals so that the VH and VL genes arerecombined onto a single replicon and are co-packaged into phagevirions. These huge libraries provide large numbers of diverseantibodies of good affinity (K_(d) ⁻¹ of about 10⁻⁸ M).

Alternatively, the repertoires may be cloned sequentially into the samevector, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA,88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g.as described in Clackson et al., Nature, 352: 624-628 (1991). PCRassembly can also be used to join VH and VL DNAs with DNA encoding aflexible peptide spacer to form single chain Fv (scFv) repertoires. Inyet another technique, “in cell PCR assembly” is used to combine VH andVL genes within lymphocytes by PCR and then clone repertoires of linkedgenes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837(1992).

The antibodies produced by naive libraries (either natural or synthetic)can be of moderate affinity (K_(d) ⁻¹ of about 10⁶ to 10⁷ M⁻¹), butaffinity maturation can also be mimicked in vitro by constructing andreselecting from secondary libraries as described in Winter et al.(1994), supra. For example, mutation can be introduced at random invitro by using error-prone polymerase (reported in Leung et al.,Technique, 1: 11-15 (1989)) in the method of Hawkins et al., J. Mol.Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl.Acad. Sci USA, 89: 3576-3580 (1992). Additionally, affinity maturationcan be performed by randomly mutating one or more CDRs, e.g. using PCRwith primers carrying random sequence spanning the CDR of interest, inselected individual Fv clones and screening for higher affinity clones.WO 9607754 (published 14 Mar. 1996) described a method for inducingmutagenesis in a complementarity determining region of an immunoglobulinlight chain to create a library of light chain genes. Another effectiveapproach is to recombine the VH or VL domains selected by phage displaywith repertoires of naturally occurring V domain variants obtained fromunimmunized donors and screen for higher affinity in several rounds ofchain reshuffling as described in Marks et al., Biotechnol., 10: 779-783(1992). This technique allows the production of antibodies and antibodyfragments with affinities in the 10⁻⁹ M range.

Nucleic acid sequence encoding a antigen can be designed using the aminoacid sequence of the desired region of antigen. Alternatively, the cDNAsequence (or fragments thereof) may be used. DNAs encoding antigen canbe prepared by a variety of methods known in the art. These methodsinclude, but are not limited to, chemical synthesis by any of themethods described in Engels et al., Agnew. Chem. Int. Ed. Engl., 28:716-734 (1989), such as the triester, phosphite, phosphoramidite andH-phosphonate methods. In one embodiment, codons preferred by theexpression host cell are used in the design of the DNA. Alternatively,DNA encoding antigen can be isolated from a genomic or cDNA library.

Following construction of the DNA molecule encoding antigen, the DNAmolecule is operably linked to an expression control sequence in anexpression vector, such as a plasmid, wherein the control sequence isrecognized by a host cell transformed with the vector. In general,plasmid vectors contain replication and control sequences which arederived from species compatible with the host cell. The vectorordinarily carries a replication site, as well as sequences which encodeproteins that are capable of providing phenotypic selection intransformed cells. Suitable vectors for expression in prokaryotic andeukaryotic host cells are known in the art and some are furtherdescribed herein. Eukaryotic organisms, such as yeasts, or cells derivedfrom multicellular organisms, such as mammals, may be used.

Optionally, the DNA encoding antigen is operably linked to a secretoryleader sequence resulting in secretion of the expression product by thehost cell into the culture medium. Examples of secretory leadersequences include stII, ecotin, lamB, herpes GD, lpp, alkalinephosphatase, invertase, and alpha factor. Also suitable for use hereinis the 36 amino acid leader sequence of protein A (Abrahmsen et al.,EMBO J., 4: 3901 (1985)).

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors of this invention andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ precipitation and electroporation. Successfultransfection is generally recognized when any indication of theoperation of this vector occurs within the host cell. Methods fortransfection are well known in the art, and some are further describedherein.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. Methods fortransformation are well known in the art, and some are further describedherein.

Prokaryotic host cells used to produce antigen can be cultured asdescribed generally in Sambrook et al., supra.

The mammalian host cells used to produce antigen can be cultured in avariety of media, which is well known in the art and some of which isdescribed herein.

The host cells referred to in this disclosure encompass cells in invitro culture as well as cells that are within a host animal.

Purification of antigen may be accomplished using art-recognizedmethods.

The purified antigen can be attached to a suitable matrix such asagarose beads, acrylamide beads, glass beads, cellulose, various acryliccopolymers, hydroxyl methacrylate gels, polyacrylic and polymethacryliccopolymers, nylon, neutral and ionic carriers, and the like, for use inthe affinity chromatographic separation of phage display clones.Attachment of the protein to the matrix can be accomplished by themethods described in Methods in Enzymology, vol. 44 (1976). A commonlyemployed technique for attaching protein ligands to polysaccharidematrices, e.g. agarose, dextran or cellulose, involves activation of thecarrier with cyanogen halides and subsequent coupling of the peptideligand's primary aliphatic or aromatic amines to the activated matrix.

Alternatively, antigen can be used to coat the wells of adsorptionplates, expressed on host cells affixed to adsorption plates or used incell sorting, or conjugated to biotin for capture withstreptavidin-coated beads, or used in any other art-known method forpanning phage display libraries.

The phage library samples are contacted with immobilized antigen underconditions suitable for binding of at least a portion of the phageparticles with the adsorbent. Normally, the conditions, including pH,ionic strength, temperature and the like are selected to mimicphysiological conditions. The phages bound to the solid phase are washedand then eluted by acid, e.g. as described in Barbas et al., Proc. Natl.Acad. Sci USA, 88: 7978-7982 (1991), or by alkali, e.g. as described inMarks et al., J. Mol. Biol., 222: 581-597 (1991), or by KLβ antigencompetition, e.g. in a procedure similar to the antigen competitionmethod of Clackson et al., Nature, 352: 624-628 (1991). Phages can beenriched 20-1,000-fold in a single round of selection. Moreover, theenriched phages can be grown in bacterial culture and subjected tofurther rounds of selection.

The efficiency of selection depends on many factors, including thekinetics of dissociation during washing, and whether multiple antibodyfragments on a single phage can simultaneously engage with antigen.Antibodies with fast dissociation kinetics (and weak binding affinities)can be retained by use of short washes, multivalent phage display andhigh coating density of antigen in solid phase. The high density notonly stabilizes the phage through multivalent interactions, but favorsrebinding of phage that has dissociated. The selection of antibodieswith slow dissociation kinetics (and good binding affinities) can bepromoted by use of long washes and monovalent phage display as describedin Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and alow coating density of antigen as described in Marks et al.,Biotechnol., 10: 779-783 (1992).

It is possible to select between phage antibodies of differentaffinities, even with affinities that differ slightly, for antigen.However, random mutation of a selected antibody (e.g. as performed insome of the affinity maturation techniques described above) is likely togive rise to many mutants, most binding to antigen, and a few withhigher affinity. With limiting antigen, rare high affinity phage couldbe competed out. To retain all the higher affinity mutants, phages canbe incubated with excess biotinylated antigen, but with the biotinylatedantigen at a concentration of lower molarity than the target molaraffinity constant for antigen. The high affinity-binding phages can thenbe captured by streptavidin-coated paramagnetic beads. Such “equilibriumcapture” allows the antibodies to be selected according to theiraffinities of binding, with sensitivity that permits isolation of mutantclones with as little as two-fold higher affinity from a great excess ofphages with lower affinity. Conditions used in washing phages bound to asolid phase can also be manipulated to discriminate on the basis ofdissociation kinetics. Anti-antigen clones may also be activityselected.

DNA encoding the hybridoma-derived monoclonal antibodies or phagedisplay Fv clones is readily isolated and sequenced using conventionalprocedures (e.g. by using oligonucleotide primers designed tospecifically amplify the heavy and light chain coding regions ofinterest from hybridoma or phage DNA template). Once isolated, the DNAcan be placed into expression vectors, which are then transfected intohost cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of the desiredmonoclonal antibodies in the recombinant host cells. Review articles onrecombinant expression in bacteria of antibody-encoding DNA includeSkerra et al., Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun,Immunol. Revs, 130: 151 (1992).

DNA encoding the Fv clones can be combined with known DNA sequencesencoding heavy chain and/or light chain constant regions (e.g. theappropriate DNA sequences can be obtained from Kabat et al., supra) toform clones encoding full or partial length heavy and/or light chains.It will be appreciated that constant regions of any isotype can be usedfor this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species. A Fv clone derived from the variable domain DNA ofone animal (such as human) species and then fused to constant region DNAof another animal species to form coding sequence(s) for “hybrid”, fulllength heavy chain and/or light chain is included in the definition of“chimeric” and “hybrid” antibody as used herein. In a preferredembodiment, a Fv clone derived from human variable DNA is fused to humanconstant region DNA to form coding sequence(s) for all human, full orpartial length heavy and/or light chains.

DNA encoding anti-antigen antibody derived from a hybridoma can also bemodified, for example, by substituting the coding sequence for humanheavy- and light-chain constant domains in place of homologous murinesequences derived from the hybridoma clone (e.g. as in the method ofMorrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). DNAencoding a hybridoma or Fv clone-derived antibody or fragment can befurther modified by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. In this manner, “chimeric” or “hybrid” antibodies areprepared that have the binding specificity of the Fv clone or hybridomaclone-derived antibodies.

Antibody Fragments

The present invention encompasses antibody fragments. In certaincircumstances there are advantages of using antibody fragments, ratherthan whole antibodies. The smaller size of the fragments allows forrapid clearance, and may lead to improved access to solid tumors.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising a salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In other embodiments, the antibody of choice is a singlechain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and sFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they are suitable for reducednonspecific binding during in vivo use. sFv fusion proteins may beconstructed to yield fusion of an effector protein at either the aminoor the carboxy terminus of an sFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example.Such linear antibody fragments may be monospecific or bispecific.

Humanized Antibodies

The present invention encompasses humanized antibodies. Various methodsfor humanizing non-human antibodies are known in the art. For example, ahumanized antibody can have one or more amino acid residues introducedinto it from a source which is non-human. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;Verhoeyen et al. (1988) Science 239:1534-1536), by substitutinghypervariable region sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567) wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some hypervariable region residuesand possibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework for the humanized antibody (Sims et al. (1993) J.Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285;Presta et al. (1993) J. Immunol., 151:2623.

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to one method, humanized antibodies areprepared by a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from therecipient and import sequences so that the desired antibodycharacteristic, such as increased affinity for the target(s), isachieved. In general, the hypervariable region residues are directly andmost substantially involved in influencing antigen binding.

Human Antibodies

Human anti-KLβ antibodies can be constructed by combining Fv clonevariable domain sequence(s) selected from human-derived phage displaylibraries with known human constant domain sequences(s) as describedabove. Alternatively, human monoclonal anti-KLβ antibodies can be madeby the hybridoma method. Human myeloma and mouse-human heteromyelomacell lines for the production of human monoclonal antibodies have beendescribed, for example, by Kozbor J. Immunol., 133: 3001 (1984); Brodeuret al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.Immunol., 147: 86 (1991).

It is now possible to produce transgenic animals (e.g. mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (JH) gene in chimeric and germ-linemutant mice results in complete inhibition of endogenous antibodyproduction. Transfer of the human germ-line immunoglobulin gene array insuch germ-line mutant mice will result in the production of humanantibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc.Natl. Acad. Sci USA, 90: 2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7: 33 (1993).

Gene shuffling can also be used to derive human antibodies fromnon-human, e.g. rodent, antibodies, where the human antibody has similaraffinities and specificities to the starting non-human antibody.According to this method, which is also called “epitope imprinting”,either the heavy or light chain variable region of a non-human antibodyfragment obtained by phage display techniques as described above isreplaced with a repertoire of human V domain genes, creating apopulation of non-human chain/human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain/humanchain chimeric scFv or Fab wherein the human chain restores the antigenbinding site destroyed upon removal of the corresponding non-human chainin the primary phage display clone, i.e. the epitope governs (imprints)the choice of the human chain partner. When the process is repeated inorder to replace the remaining non-human chain, a human antibody isobtained (see PCT WO 93/06213 published Apr. 1, 1993). Unliketraditional humanization of non-human antibodies by CDR grafting, thistechnique provides completely human antibodies, which have no FR or CDRresidues of non-human origin.

Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In one embodiment, one of the binding specificities is for KLβand the other is for any other antigen. Exemplary bispecific antibodiesmay bind to two different epitopes of the KLβ protein. Bispecificantibodies may also be used to localize cytotoxic agents to cells whichexpress KLβ. These antibodies possess a KLβ-binding arm and an arm whichbinds the cytotoxic agent (e.g. saporin, anti-interferon-α, vincaalkaloid, ricin A chain, methotrexate or radioactive isotope hapten).Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305: 537 (1983)). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 published May 13, 1993, and inTraunecker et al., EMBO J., 10: 3655 (1991).

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, CH2, and CH3 regions. Itis preferred to have the first heavy-chain constant region (CH1),containing the site necessary for light chain binding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are co-transfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach, the interface between a pair of antibodymolecules can be engineered to maximize the percentage of heterodimerswhich are recovered from recombinant cell culture. The preferredinterface comprises at least a part of the C_(H)3 domain of an antibodyconstant domain. In this method, one or more small amino acid sidechains from the interface of the first antibody molecule are replacedwith larger side chains (e.g. tyrosine or tryptophan). Compensatory“cavities” of identical or similar size to the large side chain(s) arecreated on the interface of the second antibody molecule by replacinglarge amino acid side chains with smaller ones (e.g. alanine orthreonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/00373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the HER2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (VH) connected to a light-chain variabledomain (VL) by a linker which is too short to allow pairing between thetwo domains on the same chain. Accordingly, the VH and VL domains of onefragment are forced to pair with the complementary VL and VH domains ofanother fragment, thereby forming two antigen-binding sites. Anotherstrategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See Gruber et al.,J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60(1991).

Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. The preferred dimerization domain comprises (or consistsof) an Fc region or a hinge region. In this scenario, the antibody willcomprise an Fc region and three or more antigen binding sitesamino-terminal to the Fe region. The preferred multivalent antibodyherein comprises (or consists of) three to about eight, but preferablyfour, antigen binding sites. The multivalent antibody comprises at leastone polypeptide chain (and preferably two polypeptide chains), whereinthe polypeptide chain(s) comprise two or more variable domains. Forinstance, the polypeptide chain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc,wherein VD1 is a first variable domain, VD2 is a second variable domain,Fc is one polypeptide chain of an Fc region, X1 and X2 represent anamino acid or polypeptide, and n is 0 or 1. For instance, thepolypeptide chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fcregion chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibodyherein preferably further comprises at least two (and preferably four)light chain variable domain polypeptides. The multivalent antibodyherein may, for instance, comprise from about two to about eight lightchain variable domain polypeptides. The light chain variable domainpolypeptides contemplated here comprise a light chain variable domainand, optionally, further comprise a CL domain.

Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodyare prepared by introducing appropriate nucleotide changes into theantibody nucleic acid, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of, residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution ismade to arrive at the final construct, provided that the final constructpossesses the desired characteristics. The amino acid alterations may beintroduced in the subject antibody amino acid sequence at the time thatsequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressedimmunoglobulins are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g. for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. Such altering includes deletingone or more carbohydrate moieties found in the antibody, and/or addingone or more glycosylation sites that are not present in the antibody.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. For example, antibodies with a maturecarbohydrate structure that lacks fucose attached to an Fc region of theantibody are described in US Pat Appl No US 2003/0157108 (Presta, L.).See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with abisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached toan Fc region of the antibody are referenced in WO 2003/011878,Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodieswith at least one galactose residue in the oligosaccharide attached toan Fc region of the antibody are reported in WO 1997/30087, Patel et al.See, also, WO 1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.)concerning antibodies with altered carbohydrate attached to the Fcregion thereof. See also US 2005/0123546 (Umana et al.) onantigen-binding molecules with modified glycosylation.

The preferred glycosylation variant herein comprises an Fc region,wherein a carbohydrate structure attached to the Fc region lacks fucose.Such variants have improved ADCC function. Optionally, the Fc regionfurther comprises one or more amino acid substitutions therein whichfurther improve ADCC, for example, substitutions at positions 298, 333,and/or 334 of the Fc region (Eu numbering of residues). Examples ofpublications related to “defucosylated” or “fucose-deficient” antibodiesinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; Okazaki et al. J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614(2004). Examples of cell lines producing defucosylated antibodiesinclude Lec13 CHO cells deficient in protein fucosylation (Ripka et al.Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,especially at Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells(Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004)).

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative substitutions are shownin Table 1 under the heading of “preferred substitutions”. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table 1,or as further described below in reference to amino acid classes, may beintroduced and the products screened.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine;Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

-   -   (1) hydrophobic: norleucine, met, ala, val, leu, ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: asp, glu;    -   (4) basic: his, lys, arg;    -   (5) residues that influence chain orientation: gly, pro; and    -   (6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have improved biological properties relative tothe parent antibody from which they are generated. A convenient way forgenerating such substitutional variants involves affinity maturationusing phage display. Briefly, several hypervariable region sites (e.g.6-7 sites) are mutated to generate all possible amino acid substitutionsat each site. The antibodies thus generated are displayed fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g. binding affinity) as hereindisclosed. In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and antigen. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of the immunoglobulin polypeptide, thereby generating a Fcregion variant. The Fc region variant may comprise a human Fc regionsequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprisingan amino acid modification (e.g. a substitution) at one or more aminoacid positions including that of a hinge cysteine.

In accordance with this description and the teachings of the art, it iscontemplated that in some embodiments, an antibody used in methods ofthe invention may comprise one or more alterations as compared to thewild type counterpart antibody, e.g. in the Fc region. These antibodieswould nonetheless retain substantially the same characteristics requiredfor therapeutic utility as compared to their wild type counterpart. Forexample, it is thought that certain alterations can be made in the Fcregion that would result in altered (i.e., either improved ordiminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC),e.g., as described in WO99/51642. See also Duncan & Winter Nature322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; andWO94/29351 concerning other examples of Fc region variants. WO00/42072(Presta) and WO 2004/056312 (Lowman) describe antibody variants withimproved or diminished binding to FcRs. The content of these patentpublications are specifically incorporated herein by reference. See,also, Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001). Antibodieswith increased half lives and improved binding to the neonatal Fcreceptor (FcRn), which is responsible for the transfer of maternal IgGsto the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al.,J. Immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton etal.). These antibodies comprise an Fc reg on with one or moresubstitutions therein which improve binding of the Fc region to FcRn.Polypeptide variants with altered Fc region amino acid sequences andincreased or decreased C1q binding capability are described in U.S. Pat.No. 6,194,551B1, WO99/51642. The contents of those patent publicationsare specifically incorporated herein by reference. See, also, Idusogieet al. J. Immunol. 164: 4178-4184 (2000).

Antibody Derivatives

The antibodies of the present invention can be further modified tocontain additional nonproteinaceous moieties that are known in the artand readily available. Preferably, the moieties suitable forderivatization of the antibody are water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymers are attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.

Vectors, Host Cells and Recombinant Methods

For recombinant production of an antibody, the nucleic acid encoding itis isolated and inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. DNA encoding the antibodyis readily isolated and sequenced using conventional procedures (e.g.,by using oligonucleotide probes that are capable of binding specificallyto genes encoding the heavy and light chains of the antibody). Manyvectors are available. The choice of vector depends in part on the hostcell to be used. Generally, preferred host cells are of eitherprokaryotic or eukaryotic (generally mammalian) origin. It will beappreciated that constant regions of any isotype can be used for thispurpose, including IgG, IgM, IgA, IgD, and IgE constant regions, andthat such constant regions can be obtained from any human or animalspecies.

a. Generating Antibodies Using Prokaryotic Host Cells:

-   -   i. Vector Construction

Polynucleotide sequences encoding polypeptide components of the antibodycan be obtained using standard recombinant techniques. Desiredpolynucleotide sequences may be isolated and sequenced from antibodyproducing cells such as hybridoma cells. Alternatively, polynucleotidescan be synthesized using nucleotide synthesizer or PCR techniques. Onceobtained, sequences encoding the polypeptides are inserted into arecombinant vector capable of replicating and expressing heterologouspolynucleotides in prokaryotic hosts. Many vectors that are availableand known in the art can be used for the purpose of the presentinvention. Selection of an appropriate vector will depend mainly on thesize of the nucleic acids to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components, depending on its function (amplification orexpression of heterologous polynucleotide, or both) and itscompatibility with the particular host cell in which it resides. Thevector components generally include, but are not limited to: an originof replication, a selection marker gene, a promoter, a ribosome bindingsite (RBS), a signal sequence, the heterologous nucleic acid insert anda transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

The expression vector may comprise two or more promoter-cistron pairs,encoding each of the polypeptide components. A promoter is anuntranslated regulatory sequence located upstream (5′) to a cistron thatmodulates its expression. Prokaryotic promoters typically fall into twoclasses, inducible and constitutive. Inducible promoter is a promoterthat initiates increased levels of transcription of the cistron underits control in response to changes in the culture condition, e.g. thepresence or absence of a nutrient or a change in temperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector. Both the native promotersequence and many heterologous promoters may be used to directamplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to cistrons encoding the target light andheavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In one aspect, each cistron within the recombinant vector comprises asecretion signal sequence component that directs translocation of theexpressed polypeptides across a membrane. In general, the signalsequence may be a component of the vector, or it may be a part of thetarget polypeptide DNA that is inserted into the vector. The signalsequence selected for the purpose of this invention should be one thatis recognized and processed (i.e. cleaved by a signal peptidase) by thehost cell. For prokaryotic host cells that do not recognize and processthe signal sequences native to the heterologous polypeptides, the signalsequence is substituted by a prokaryotic signal sequence selected, forexample, from the group consisting of the alkaline phosphatase,penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB,PhoE, PelB, OmpA and MBP. In one embodiment, the signal sequences usedin both cistrons of the expression system are STII signal sequences orvariants thereof.

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. coli trxB−strains) provide cytoplasm conditions that are favorable for disulfidebond formation, thereby permitting proper folding and assembly ofexpressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

Prokaryotic host cells suitable for expressing antibodies includeArchaebacteria and Eubacteria, such as Gram-negative or Gram-positiveorganisms. Examples of useful bacteria include Escherichia (e.g., E.coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas species(e.g., P. aeruginosa), Salmonella typhimurium, Serratia marcescans,Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or Paracoccus. Inone embodiment, gram-negative cells are used. In one embodiment, E. colicells are used as hosts for the invention. Examples of E. coli strainsinclude strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2(Washington, D.C.: American Society for Microbiology, 1987), pp.1190-1219; ATCC Deposit No. 27,325) and derivatives thereof, includingstrain 33D3 having genotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8ΔompTΔ(nmpc-fepE) degP41 kanR (U.S. Pat. No. 5,639,635). Other strainsand derivatives thereof, such as E. coli 294 (ATCC 31,446), E. coli B,E. coli, 1776 (ATCC 31,537) and E. coli RV308 (ATCC 31,608) are alsosuitable. These examples are illustrative rather than limiting. Methodsfor constructing derivatives of any of the above-mentioned bacteriahaving defined genotypes are known in the art and described in, forexample, Bass et al., Proteins, 8:309-314 (1990). It is generallynecessary to select the appropriate bacteria taking into considerationreplicability of the replicon in the cells of a bacterium. For example,E. coli, Serratia, or Salmonella species can be suitably used as thehost when well known plasmids such as pBR322, pBR325, pACYC177, orpKN410 are used to supply the replicon. Typically the host cell shouldsecrete minimal amounts of proteolytic enzymes, and additional proteaseinhibitors may desirably be incorporated in the cell culture.

ii. Antibody Production

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides are grown in mediaknown in the art and suitable for culture of the selected host cells.Examples of suitable media include luria broth (LB) plus necessarynutrient supplements. In some embodiments, the media also contains aselection agent, chosen based on the construction of the expressionvector, to selectively permit growth of prokaryotic cells containing theexpression vector. For example, ampicillin is added to media for growthof cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the preferred temperature ranges from about20° C. to about 39° C., more preferably from about 25° C. to about 37°C., even more preferably at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH is preferably from about 6.8 to about 7.4,and more preferably about 7.0.

If an inducible promoter is used in the expression vector, proteinexpression is induced under conditions suitable for the activation ofthe promoter. In one aspect, PhoA promoters are used for controllingtranscription of the polypeptides. Accordingly, the transformed hostcells are cultured in a phosphate-limiting medium for induction.Preferably, the phosphate-limiting medium is the C.R.A.P medium (see,e.g., Simmons et al., J. Immunol. Methods (2002), 263:133-147). Avariety of other inducers may be used, according to the vector constructemployed, as is known in the art.

In one embodiment, the expressed polypeptides of the present inventionare secreted into and recovered from the periplasm of the host cells.Protein recovery typically involves disrupting the microorganism,generally by such means as osmotic shock, sonication or lysis. Oncecells are disrupted, cell debris or whole cells may be removed bycentrifugation or filtration. The proteins may be further purified, forexample, by affinity resin chromatography. Alternatively, proteins canbe transported into the culture media and isolated therein. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as polyacrylamide gel electrophoresis (PAGE)and Western blot assay.

In one aspect, antibody production is conducted in large quantity by afermentation process. Various large-scale fed-batch fermentationprocedures are available for production of recombinant proteins.Large-scale fermentations have at least 1000 liters of capacity,preferably about 1,000 to 100,000 liters of capacity. These fermentorsuse agitator impellers to distribute oxygen and nutrients, especiallyglucose (the preferred carbon/energy source). Small scale fermentationrefers generally to fermentation in a fermentor that is no more thanapproximately 100 liters in volumetric capacity, and can range fromabout 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD550 of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides, variousfermentation conditions can be modified. For example, to improve theproper assembly and folding of the secreted antibody polypeptides,additional vectors overexpressing chaperone proteins, such as Dsbproteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolylcis,trans-isomerase with chaperone activity) can be used to co-transformthe host prokaryotic cells. The chaperone proteins have beendemonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.(1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S.Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72(1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system.

iii. Antibody Purification

Standard protein purification methods known in the art can be employed.The following procedures are exemplary of suitable purificationprocedures: fractionation on immunoaffinity or ion-exchange columns,ethanol precipitation, reverse phase HPLC, chromatography on silica oron a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE,ammonium sulfate precipitation, and gel filtration using, for example,Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the full length antibody products.Protein A is a 41 kD cell wall protein from Staphylococcus aureas whichbinds with a high affinity to the Fc region of antibodies. Lindmark etal (1983) J. Immunol. Meth. 62:1-13. The solid phase to which Protein Ais immobilized is preferably a column comprising a glass or silicasurface, more preferably a controlled pore glass column or a silicicacid column. In some applications, the column has been coated with areagent, such as glycerol, in an attempt to prevent nonspecificadherence of contaminants.

As the first step of purification, the preparation derived from the cellculture as described above is applied onto the Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase is then washed to remove contaminantsnon-specifically bound to the solid phase. Finally the antibody ofinterest is recovered from the solid phase by elution.

b. Generating Antibodies Using Eukaryotic Host Cells:

The vector components generally include, but are not limited to, one ormore of the following: a signal sequence, an origin of replication, oneor more marker genes, an enhancer element, a promoter, and atranscription termination sequence.

-   -   (i) Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

(ii) Origin of Replication

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used only because it contains the early promoter.

(iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, where relevant, or (c) supply critical nutrients notavailable from complex media.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCCCRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the antibodypolypeptide nucleic acid. Promoter sequences are known for eukaryotes.Virtually alleukaryotic genes have an AT-rich region locatedapproximately 25 to 30 bases upstream from the site where transcriptionis initiated. Another sequence found 70 to 80 bases upstream from thestart of transcription of many genes is a CNCAAT region where N may beany nucleotide. At the 3′ end of most eukaryotic genes is an AATAAAsequence that may be the signal for addition of the poly A tail to the3′ end of the coding sequence. All of these sequences are suitablyinserted into eukaryotic expression vectors.

Antibody polypeptide transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. Alternatively, the Rous Sarcoma Virus long terminal repeatcan be used as the promoter.

(v) Enhancer Element Component

Transcription of DNA encoding the antibody polypeptide of this inventionby higher eukaryotes is often increased by inserting an enhancersequence into the vector. Many enhancer sequences are now known frommammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theantibody polypeptide-encoding sequence, but is preferably located at asite 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells will typically alsocontain sequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding an antibody. One useful transcription termination component isthe bovine growth hormone polyadenylation region. See WO94/11026 and theexpression vector disclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include higher eukaryote cells described herein, includingvertebrate host cells. Propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(viii) Culturing the Host Cells

The host cells used to produce an antibody may be cultured in a varietyof media. Commercially available media such as Ham's F10 (Sigma),Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), andDulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable forculturing the host cells. In addition, any of the media described in Hamet al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255(1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re. 30,985 may be usedas culture media for the host cells. Any of these media may besupplemented as necessary with hormones and/or other growth factors(such as insulin, transferrin, or epidermal growth factor), salts (suchas sodium chloride, calcium, magnesium, and phosphate), buffers (such asHEPES), nucleotides (such as adenosine and thymidine), antibiotics (suchas GENTAMYCIN™ drug), trace elements (defined as inorganic compoundsusually present at final concentrations in the micromolar range), andglucose or an equivalent energy source. Any other necessary supplementsmay also be included at appropriate concentrations that would be knownto those skilled in the art. The culture conditions, such astemperature, pH, and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

(ix) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, or directly secreted into the medium. If the antibodyis produced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, are removed, for example, bycentrifugation or ultrafiltration. Where the antibody is secreted intothe medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a CH3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Immunoconjugates

The invention also provides immunoconjugates (interchangeably termed“antibody-drug conjugates” or “ADC”), comprising any of the anti-KLβantibodies described herein conjugated to a cytotoxic agent such as achemotherapeutic agent, a drug, a growth inhibitory agent, a toxin(e.g., an enzymatically active toxin of bacterial, fungal, plant, oranimal origin, or fragments thereof), or a radioactive isotope (i.e., aradioconjugate).

The use of antibody-drug conjugates for the local delivery of cytotoxicor cytostatic agents, i.e. drugs to kill or inhibit tumor cells in thetreatment of cancer (Syrigos and Epenetos (1999) Anticancer Research19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg Del. Rev.26:151-172; U.S. Pat. No. 4,975,278) allows targeted delivery of thedrug moiety to tumors, and intracellular accumulation therein, wheresystemic administration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells as well as the tumorcells sought to be eliminated (Baldwin et al., (1986) Lancet pp. (Mar.15, 1986):603-05; Thorpe, (1985) “Antibody Carriers Of Cytotoxic AgentsIn Cancer Therapy: A Review,” in Monoclonal Antibodies '84: BiologicalAnd Clinical Applications, A. Pinchera et al. (ed.s), pp. 475-506).Maximal efficacy with minimal toxicity is sought thereby. Bothpolyclonal antibodies and monoclonal antibodies have been reported asuseful in these strategies (Rowland et al., (1986) Cancer Immunol.Immunother., 21:183-87). Drugs used in these methods include daunomycin,doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra).Toxins used in antibody-toxin conjugates include bacterial toxins suchas diphtheria toxin, plant toxins such as ricin, small molecule toxinssuch as geldanamycin (Mandler et al (2000) Jour. of the Nat. CancerInst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med. Chem.Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998)Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342). Thetoxins may effect their cytotoxic and cytostatic effects by mechanismsincluding tubulin binding, DNA binding, or topoisomerase inhibition.Some cytotoxic drugs tend to be inactive or less active when conjugatedto large antibodies or protein receptor ligands.

ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotopeconjugate composed of a murine IgG1 kappa monoclonal antibody directedagainst the CD20 antigen found on the surface of normal and malignant Blymphocytes and ¹¹¹In or ⁹⁰Y radioisotope bound by a thiourealinker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med.27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al(2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al (2002) J. Clin.Oncol. 20(15):3262-69). Although ZEVALIN has activity against B-cellnon-Hodgkin's Lymphoma (NHL), administration results in severe andprolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab ozogamicin,Wyeth Pharmaceuticals), an antibody drug conjugate composed of a hu CD33antibody linked to calicheamicin, was approved in 2000 for the treatmentof acute myeloid leukemia by injection (Drugs of the Future (2000)25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040;5,693,762; 5,739,116; 5,767,285; 5,773,001). Cantuzumab mertansine(Immunogen, Inc.), an antibody drug conjugate composed of the huC242antibody linked via the disulfide linker SPP to the maytansinoid drugmoiety, DM1, is advancing into Phase II trials for the treatment ofcancers that express CanAg, such as colon, pancreatic, gastric, andothers. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), anantibody drug conjugate composed of the anti-prostate specific membraneantigen (PSMA) monoclonal antibody linked to the maytansinoid drugmoiety, DM1, is under development for the potential treatment ofprostate tumors. The auristatin peptides, auristatin E (AE) andmonomethylauristatin (MMAE), synthetic analogs of dolastatin, wereconjugated to chimeric monoclonal antibodies cBR96 (specific to Lewis Yon carcinomas) and cAC10 (specific to CD30 on hematologicalmalignancies) (Doronina et al (2003) Nature Biotechnology 21(7):778-784)and are under therapeutic development.

Chemotherapeutic agents useful in the generation of immunoconjugates aredescribed herein (eg., above). Enzymatically active toxins and fragmentsthereof that can be used include diphtheria A chain, nonbinding activefragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.See, e.g., WO 93/21232 published Oct. 28, 1993. A variety ofradionuclides are available for the production of radioconjugatedantibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, dolastatins, aurostatins, atrichothecene, and CC1065, and the derivatives of these toxins that havetoxin activity, are also contemplated herein.

i. Maytansine and Maytansinoids

In some embodiments, the immunoconjugate comprises an antibody (fulllength or fragments) conjugated to one or more maytansinoid molecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533.

Maytansinoid drug moieties are attractive drug moieties in antibody drugconjugates because they are: (i) relatively accessible to prepare byfermentation or chemical modification, derivatization of fermentationproducts, (ii) amenable to derivatization with functional groupssuitable for conjugation through the non-disulfide linkers toantibodies, (iii) stable in plasma, and (iv) effective against a varietyof tumor cell lines.

Immunoconjugates containing maytansinoids, methods of making same, andtheir therapeutic use are disclosed, for example, in U.S. Pat. Nos.5,208,020, 5,416,064 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) describedimmunoconjugates comprising a maytansinoid designated DM1 linked to themonoclonal antibody C242 directed against human colorectal cancer. Theconjugate was found to be highly cytotoxic towards cultured colon cancercells, and showed antitumor activity in an in vivo tumor growth assay.Chari et al., Cancer Research 52:127-131 (1992) describeimmunoconjugates in which a maytansinoid was conjugated via a disulfidelinker to the murine antibody A7 binding to an antigen on human coloncancer cell lines, or to another murine monoclonal antibody TA.1 thatbinds the HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansinoidconjugate was tested in vitro on the human breast cancer cell lineSK-BR-3, which expresses 3×10⁵ HER-2 surface antigens per cell. The drugconjugate achieved a degree of cytotoxicity similar to the freemaytansinoid drug, which could be increased by increasing the number ofmaytansinoid molecules per antibody molecule. The A7-maytansinoidconjugate showed low systemic cytotoxicity in mice.

Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. See, e.g., U.S. Pat. No. 5,208,020 (the disclosure of which ishereby expressly incorporated by reference). An average of 3-4maytansinoid molecules conjugated per antibody molecule has shownefficacy in enhancing cytotoxicity of target cells without negativelyaffecting the function or solubility of the antibody, although even onemolecule of toxin/antibody would be expected to enhance cytotoxicityover the use of naked antibody. Maytansinoids are well known in the artand can be synthesized by known techniques or isolated from naturalsources. Suitable maytansinoids are disclosed, for example, in U.S. Pat.No. 5,208,020 and in the other patents and nonpatent publicationsreferred to hereinabove. Preferred maytansinoids are maytansinol andmaytansinol analogues modified in the aromatic ring or at otherpositions of the maytansinol molecule, such as various maytansinolesters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, Chari etal., Cancer Research 52:127-131 (1992), and U.S. patent application Ser.No. 10/960,602, filed Oct. 8, 2004, the disclosures of which are herebyexpressly incorporated by reference. Antibody-maytansinoid conjugatescomprising the linker component SMCC may be prepared as disclosed inU.S. patent application Ser. No. 10/960,602, filed Oct. 8, 2004. Thelinking groups include disulfide groups, thioether groups, acid labilegroups, photolabile groups, peptidase labile groups, or esterase labilegroups, as disclosed in the above-identified patents, disulfide andthioether groups being preferred. Additional linking groups aredescribed and exemplified herein.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlssonet al., Biochem. J. 173:723-737 (1978)) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

ii. Auristatins and Dolastatins

In some embodiments, the immunoconjugate comprises an antibodyconjugated to dolastatins or dolostatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in“Monomethylvaline Compounds Capable of Conjugation to Ligands”, U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the disclosure of which isexpressly incorporated by reference in its entirety.

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schroder and K. Lübke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. No. 5,635,483; U.S.Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465;Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R.,et al. Synthesis, 1996, 719-725; and Pettit et al (1996) J. Chem. Soc.Perkin Trans. 1 5:859-863. See also Doronina (2003) Nat Biotechnol21(7):778-784; “Monomethylvaline Compounds Capable of Conjugation toLigands”, U.S. Ser. No. 10/983,340, filed Nov. 5, 2004, herebyincorporated by reference in its entirety (disclosing, e.g., linkers andmethods of preparing monomethylvaline compounds such as MMAE and MMAFconjugated to linkers).

iii. Calicheamicin

In other embodiments, the immunoconjugate comprises an antibodyconjugated to one or more calicheamicin molecules. The calicheamicinfamily of antibiotics are capable of producing double-stranded DNAbreaks at sub-picomolar concentrations. For the preparation ofconjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001,5,877,296 (all to American Cyanamid Company). Structural analogues ofcalicheamicin which may be used include, but are not limited to, γ₁^(I), α₂ ^(I), α₃ ^(I), N-acetyl-γ₁ ^(I), PSAG and θ^(I) ₁ (Hinman etal., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research58:2925-2928 (1998) and the aforementioned U.S. patents to AmericanCyanamid). Another anti-tumor drug that the antibody can be conjugatedis QFA which is an antifolate. Both calicheamicin and QFA haveintracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

iv. Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies includeBCNU, streptozoicin, vincristine and 5-fluorouracil, the family ofagents known collectively LL-E33288 complex described in U.S. Pat. Nos.5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt²¹¹, ¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu. When the conjugate is used for detection, itmay comprise a radioactive atom for scintigraphic studies, for exampletc^(99m) or I¹²³, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The compounds expressly contemplate, but are not limited to, ADCprepared with cross-linker reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC,MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A). Seepages 467-498, 2003-2004 Applications Handbook and Catalog.

v. Preparation of Antibody Drug Conjugates

In the antibody drug conjugates (ADC), an antibody (Ab) is conjugated toone or more drug moieties (D), e.g. about 1 to about 20 drug moietiesper antibody, through a linker (L). The ADC of Formula I may be preparedby several routes, employing organic chemistry reactions, conditions,and reagents known to those skilled in the art, including: (1) reactionof a nucleophilic group of an antibody with a bivalent linker reagent,to form Ab-L, via a covalent bond, followed by reaction with a drugmoiety D; and (2) reaction of a nucleophilic group of a drug moiety witha bivalent linker reagent, to form D-L, via a covalent bond, followed byreaction with the nucleophilic group of an antibody. Additional methodsfor preparing ADC are described herein.

Ab-(L-D)_(p)  I

The linker may be composed of one or more linker components. Exemplarylinker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl(“MP”), valine-citrulline (“val-cit”), alanine-phenylalanine(“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), N-Succinimidyl4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”), andN-Succinimidyl(4-iodo-acetyl)aminobenzoate (“SIAB”). Additional linkercomponents are known in the art and some are described herein. See also“Monomethylvaline Compounds Capable of Conjugation to Ligands”, U.S.Ser. No. 10/983,340, filed Nov. 5, 2004, the contents of which arehereby incorporated by reference in its entirety.

In some embodiments, the linker may comprise amino acid residues.Exemplary amino acid linker components include a dipeptide, atripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptidesinclude: valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acid analogs, such as citrulline. Amino acid linkercomponents can be designed and optimized in their selectivity forenzymatic cleavage by a particular enzymes, for example, atumor-associated protease, cathepsin B, C and D, or a plasmin protease.

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibody(or fragment thereof) by introducing one, two, three, four, or morecysteine residues (e.g., preparing mutant antibodies comprising one ormore non-native cysteine amino acid residues).

Antibody drug conjugates may also be produced by modification of theantibody to introduce electrophilic moieties, which can react withnucleophilic substituents on the linker reagent or drug. The sugars ofglycosylated antibodies may be oxidized, e.g. with periodate oxidizingreagents, to form aldehyde or ketone groups which may react with theamine group of linker reagents or drug moieties. The resulting imineSchiff base groups may form a stable linkage, or may be reduced, e.g. byborohydride reagents to form stable amine linkages. In one embodiment,reaction of the carbohydrate portion of a glycosylated antibody witheither glactose oxidase or sodium meta-periodate may yield carbonyl(aldehyde and ketone) groups in the protein that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, proteins containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Suchaldehyde can be reacted with a drug moiety or linker nucleophile.

Likewise, nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.The length of DNA may comprise respective regions encoding the twoportions of the conjugate either adjacent one another or separated by aregion encoding a linker peptide which does not destroy the desiredproperties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin)which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

Binding Oligopeptides

Binding oligopeptides are oligopeptides that bind, preferablyspecifically, to KLβ, FGFR or KLβ-FGFR complex as described herein.Binding oligopeptides may be chemically synthesized using knownoligopeptide synthesis methodology or may be prepared and purified usingrecombinant technology. Binding oligopeptides are usually at least about5 amino acids in length, alternatively at least about 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100 amino acids in length or more, wherein such oligopeptides that arecapable of binding, preferably specifically, to a polypeptide asdescribed herein. Binding oligopeptides may be identified without undueexperimentation using well known techniques. In this regard, it is notedthat techniques for screening oligopeptide libraries for oligopeptidesthat are capable of specifically binding to a polypeptide target arewell known in the art (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373,4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCTPublication Nos. WO 84/03506 and WO84/03564; Geysen et al., Proc. Natl.Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad.Sci. U.S.A., 82:178-182 (1985); Geysen et al., in Synthetic Peptides asAntigens, 130-149 (1986); Geysen et al., J. Immunol. Meth., 102:259-274(1987); Schoofs et al., J. Immunol., 140:611-616 (1988), Cwirla, S. E.et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378; Lowman, H. B. et al.(1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352:624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. etal. (1991) Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991)Current Opin. Biotechnol., 2:668).

In this regard, bacteriophage (phage) display is one well knowntechnique which allows one to screen large oligopeptide libraries toidentify member(s) of those libraries which are capable of specificallybinding to a polypeptide target. Phage display is a technique by whichvariant polypeptides are displayed as fusion proteins to the coatprotein on the surface of bacteriophage particles (Scott, J. K. andSmith, G. P. (1990) Science, 249: 386). The utility of phage displaylies in the fact that large libraries of selectively randomized proteinvariants (or randomly cloned cDNAs) can be rapidly and efficientlysorted for those sequences that bind to a target molecule with highaffinity. Display of peptide (Cwirla, S. E. et al. (1990) Proc. Natl.Acad. Sci. USA, 87:6378) or protein (Lowman, H. B. et al. (1991)Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624;Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al.(1991) Proc. Natl. Acad. Sci. USA, 88:8363) libraries on phage have beenused for screening millions of polypeptides or oligopeptides for oneswith specific binding properties (Smith, G. P. (1991) Current Opin.Biotechnol., 2:668). Sorting phage libraries of random mutants requiresa strategy for constructing and propagating a large number of variants,a procedure for affinity purification using the target receptor, and ameans of evaluating the results of binding enrichments. U.S. Pat. Nos.5,223,409, 5,403,484, 5,571,689, and 5,663,143.

Although most phage display methods have used filamentous phage,lambdoid phage display systems (WO 95/34683; U.S. Pat. No. 5,627,024),T4 phage display systems (Ren et al., Gene, 215: 439 (1998); Zhu et al.,Cancer Research, 58(15): 3209-3214 (1998); Jiang et al., Infection &Immunity, 65(11): 4770-4777 (1997); Ren et al., Gene, 195(2):303-311(1997); Ren, Protein Sci., 5: 1833 (1996); Efimov et al., Virus Genes,10: 173 (1995)) and T7 phage display systems (Smith and Scott, Methodsin Enzymology, 217: 228-257 (1993); U.S. Pat. No. 5,766,905) are alsoknown.

Many other improvements and variations of the basic phage displayconcept have now been developed. These improvements enhance the abilityof display systems to screen peptide libraries for binding to selectedtarget molecules and to display functional proteins with the potentialof screening these proteins for desired properties. Combinatorialreaction devices for phage display reactions have been developed (WO98/14277) and phage display libraries have been used to analyze andcontrol bimolecular interactions (WO 98/20169; WO 98/20159) andproperties of constrained helical peptides (WO 98/20036). WO 97/35196describes a method of isolating an affinity ligand in which a phagedisplay library is contacted with one solution in which the ligand willbind to a target molecule and a second solution in which the affinityligand will not bind to the target molecule, to selectively isolatebinding ligands. WO 97/46251 describes a method of biopanning a randomphage display library with an affinity purified antibody and thenisolating binding phage, followed by a micropanning process usingmicroplate wells to isolate high affinity binding phage. The use ofStaphlylococcus aureus protein A as an affinity tag has also beenreported (Li et al. (1998) Mol Biotech., 9:187). WO 97/47314 describesthe use of substrate subtraction libraries to distinguish enzymespecificities using a combinatorial library which may be a phage displaylibrary. A method for selecting enzymes suitable for use in detergentsusing phage display is described in WO 97/09446. Additional methods ofselecting specific binding proteins are described in U.S. Pat. Nos.5,498,538, 5,432,018, and WO 98/15833.

Methods of generating peptide libraries and screening these librariesare also disclosed in U.S. Pat. Nos. 5,723,286, 5,432,018, 5,580,717,5,427,908, 5,498,530, 5,770,434, 5,734,018, 5,698,426, 5,763,192, and5,723,323.

Binding Small Molecules

Binding small molecules are preferably organic molecules other thanoligopeptides or antibodies as defined herein that bind, preferablyspecifically, to KLβ, FGFR or KLβ/FGFR complex as described herein.Binding organic small molecules may be identified and chemicallysynthesized using known methodology (see, e.g., PCT Publication Nos.WO00/00823 and WO00/39585). Binding organic small molecules are usuallyless than about 2000 daltons in size, alternatively less than about1500, 750, 500, 250 or 200 daltons in size, wherein such organic smallmolecules that are capable of binding, preferably specifically, to apolypeptide as described herein may be identified without undueexperimentation using well known techniques. In this regard, it is notedthat techniques for screening organic small molecule libraries formolecules that are capable of binding to a polypeptide target are wellknown in the art (see, e.g., PCT Publication Nos. WO00/00823 andWO00/39585). Binding organic small molecules may be, for example,aldehydes, ketones, oximes, hydrazones, semicarbazones, carbazides,primary amines, secondary amines, tertiary amines, N-substitutedhydrazines, hydrazides, alcohols, ethers, thiols, thioethers,disulfides, carboxylic acids, esters, amides, ureas, carbamates,carbonates, ketals, thioketals, acetals, thioacetals, aryl halides, arylsulfonates, alkyl halides, alkyl sulfonates, aromatic compounds,heterocyclic compounds, anilines, alkenes, alkynes, diols, aminoalcohols, oxazolidines, oxazolines, thiazolidines, thiazolines,enamines, sulfonamides, epoxides, aziridines, isocyanates, sulfonylchlorides, diazo compounds, acid chlorides, or the like.

Screening for Antibodies, Oligopeptides and Organic Small Molecules withDesired Properties

In some embodiments, the antagonists bind KLβ, and in some embodiments,may modulate one or more aspects of KLβ-associated effects, includingbut not limited to may modulate one or more aspects of KLβ-associatedeffects, including but not limited to binding FGFR4 (optionally inconjunction with heparin), binding FGF19 (optionally in conjunction withheparin), binding FGFR4 and FGF19 (optionally in conjunction withheparin), promoting FGF19-mediated induction of cFos, Junb and/or Junc(in vitro or in vivo), promoting FGFR4 and/or FGF19 down streamsignaling (including but not limited to FRS2 phosphorylation, ERK1/2phosphorylation and Wnt pathway activation), and/or promotion of anybiologically relevant KLβ and/or FGFR4 biological pathway, and/orpromotion of a tumor, cell proliferative disorder or a cancer; and/orpromotion of a disorder associated with KLβ expression and/or activity(such as increased KLβ expression and/or activity).

The purified antibodies can be further characterized by a series ofassays including, but not limited to, N-terminal sequencing, amino acidanalysis, non-denaturing size exclusion high pressure liquidchromatography (HPLC), mass spectrometry, ion exchange chromatographyand papain digestion.

In certain embodiments of the invention, the antibodies produced hereinare analyzed for their biological activity. In some embodiments, theantibodies of the present invention are tested for their antigen bindingactivity. The antigen binding assays that are known in the art and canbe used herein include without limitation any direct or competitivebinding assays using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, fluorescent immunoassays, andprotein A immunoassays. Illustrative antigen binding assay are providedbelow in the Examples section.

Anti-KLβ antibodies possessing the properties described herein can beobtained by screening anti-KLβ hybridoma clones for the desiredproperties by any convenient method.

Other functional assays to determine the binding capacity of anti-KLβantibodies are known in the art, some of which are exemplified herein.

Anti-FGFR4 antibodies possessing the properties described herein can beobtained by screening anti-FGFR4 hybridoma clones for the desiredproperties by any convenient method.

Other functional assays to determine the binding capacity of anti-FGFR4antibodies are known in the art, some of which are exemplified herein.

To screen for antibodies, oligopeptides or other organic small moleculeswhich bind to an epitope on a polypeptide bound by an antibody ofinterest, a routine cross-blocking assay such as that described inAntibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, EdHarlow and David Lane (1988), can be performed. This assay can be usedto determine if a test antibody, oligopeptide or other organic smallmolecule binds the same site or epitope as a known antibody.Alternatively, or additionally, epitope mapping can be performed bymethods known in the art. For example, the antibody sequence can bemutagenized such as by alanine scanning, to identify contact residues.The mutant antibody is initially tested for binding with polyclonalantibody to ensure proper folding. In a different method, peptidescorresponding to different regions of a polypeptide can be used incompetition assays with the test antibodies or with a test antibody andan antibody with a characterized or known epitope.

In some embodiments, the present invention contemplates alteredantibodies that possess some but not all effector functions, which makeit a desired candidate for many applications in which the half life ofthe antibody in vivo is important yet certain effector functions (suchas complement and ADCC) are unnecessary or deleterious. In certainembodiments, the Fc activities of the produced immunoglobulin aremeasured to ensure that only the desired properties are maintained. Invitro and/or in vivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu Rev. Immunol 9:457-92 (1991). Anexample of an in vitro assay to assess ADCC activity of a molecule ofinterest is described in U.S. Pat. No. 5,500,362 or 5,821,337. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.PNAS (USA) 95:652-656 (1998). C1q binding assays may also be carried outto confirm that the antibody is unable to bind C1q and hence lacks CDCactivity. To assess complement activation, a CDC assay, e.g. asdescribed in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996),may be performed. FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art.

In some embodiments, altered antibodies that possess increased effectorfunctions and/or increased half-life are useful.

Polypeptides and Nucleic Acids

Nucleotide sequences have various applications in the art of molecularbiology, as well as uses for therapy, etc. Polypeptide-encoding nucleicacid will also be useful for the preparation of polypeptides by therecombinant techniques described herein, wherein those polypeptides mayfind use, for example, in the preparation of antibodies as describedherein.

A full-length native sequence polypeptide gene, or portions thereof, maybe used as hybridization probes for a cDNA library to isolate othercDNAs (for instance, those encoding naturally-occurring variants of apolypeptide or a polypeptide from other species) which have a desiredsequence identity to a native polypeptide sequence disclosed herein.Optionally, the length of the probes will be about 20 to about 50 bases.The hybridization probes may be derived from at least partially novelregions of the full length native nucleotide sequence wherein thoseregions may be determined without undue experimentation or from genomicsequences including promoters, enhancer elements and introns of nativesequence polypeptide. By way of example, a screening method willcomprise isolating the coding region of the polypeptide gene using theknown DNA sequence to synthesize a selected probe of about 40 bases.Hybridization probes may be labeled by a variety of labels, includingradionucleotides such as ³²P or ³⁵S, or enzymatic labels such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems. Labeled probes having a sequence complementary to that of thepolypeptide gene of the present invention can be used to screenlibraries of human cDNA, genomic DNA or mRNA to determine which membersof such libraries the probe hybridizes to. Hybridization techniques aredescribed in further detail in the Examples below. Any EST sequencesdisclosed in the present application may similarly be employed asprobes, using the methods disclosed herein.

Other useful fragments of the polypeptide-encoding nucleic acids includeantisense or sense oligonucleotides comprising a singe-stranded nucleicacid sequence (either RNA or DNA) capable of binding to target apolypeptide mRNA (sense) or a polypeptide DNA (antisense) sequence.Antisense or sense oligonucleotides, according to the present invention,comprise a fragment of the coding region of a DNA encoding hepsin,pro-HGF or binding fragments as described herein. Such a fragmentgenerally comprises at least about 14 nucleotides, preferably from about14 to 30 nucleotides. The ability to derive an antisense or a senseoligonucleotide, based upon a cDNA sequence encoding a given protein isdescribed in, for example, Stein and Cohen (Cancer Res. 48:2659, 1988)and van der Krol et al. (BioTechniques 6:958, 1988).

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block transcriptionor translation of the target sequence by one of several means, includingenhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. Such methods areencompassed by the present invention. The antisense oligonucleotidesthus may be used to block expression of a protein, wherein the proteinmay play a role in the induction of cancer in mammals. Antisense orsense oligonucleotides further comprise oligonucleotides having modifiedsugar-phosphodiester backbones (or other sugar linkages, such as thosedescribed in WO 91/06629) and wherein such sugar linkages are resistantto endogenous nucleases. Such oligonucleotides with resistant sugarlinkages are stable in vivo (i.e., capable of resisting enzymaticdegradation) but retain sequence specificity to be able to bind totarget nucleotide sequences.

Preferred intragenic sites for antisense binding include the regionincorporating the translation initiation/start codon (5′-AUG/5′-ATG) ortermination/stop codon (5′-UAA, 5′-UAG and 5-UGA/5′-TAA, 5′-TAG and5′-TGA) of the open reading frame (ORF) of the gene. These regions referto a portion of the mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation initiation or termination codon. Other preferred regions forantisense binding include: introns; exons; intron-exon junctions; theopen reading frame (ORF) or “coding region,” which is the region betweenthe translation initiation codon and the translation termination codon;the 5′ cap of an mRNA which comprises an N7-methylated guanosine residuejoined to the 5′-most residue of the mRNA via a 5′-5′ triphosphatelinkage and includes 5′ cap structure itself as well as the first 50nucleotides adjacent to the cap; the 5′ untranslated region (5′UTR), theportion of an mRNA in the 5′ direction from the translation initiationcodon, and thus including nucleotides between the 5′ cap site and thetranslation initiation codon of an mRNA or corresponding nucleotides onthe gene; and the 3′ untranslated region (3′UTR), the portion of an mRNAin the 3′ direction from the translation termination codon, and thusincluding nucleotides between the translation termination codon and 3′end of an mRNA or corresponding nucleotides on the gene.

Specific examples of preferred antisense compounds useful for inhibitingexpression of a polypeptide include oligonucleotides containing modifiedbackbones or non-natural internucleoside linkages. Oligonucleotideshaving modified backbones include those that retain a phosphorus atom inthe backbone and those that do not have a phosphorus atom in thebackbone. For the purposes of this specification, and as sometimesreferenced in the art, modified oligonucleotides that do not have aphosphorus atom in their internucleoside backbone can also be consideredto be oligonucleosides. Preferred modified oligonucleotide backbonesinclude, for example, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included. Representative UnitedStates patents that teach the preparation of phosphorus-containinglinkages include, but are not limited to, U.S. Pat. Nos. 3,687,808;4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599;5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, each of whichis herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH.sub.2 component parts. RepresentativeUnited States patents that teach the preparation of sucholigonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, each of whichis herein incorporated by reference.

In other preferred antisense oligonucleotides, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Preferred antisense oligonucleotides incorporate phosphorothioatebackbones and/or heteroatom backbones, and in particular —CH₂—NH—O—CH₂—,—CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone],—CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂—[wherein the native phosphodiester backbone is represented as—O—P—O—CH₂—] described in the above referenced U.S. Pat. No. 5,489,677,and the amide backbones of the above referenced U.S. Pat. No. 5,602,240.Also preferred are antisense oligonucleotides having morpholino backbonestructures of the above-referenced U.S. Pat. No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O-alkyl, S-alkyl, or N-alkyl; O-alkenyl,S-alkeynyl, or N-alkenyl; O-alkynyl, S-alkynyl or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred antisense oligonucleotides comprise one of the followingat the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl,alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃,OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂ CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂).

A further preferred modification includes Locked Nucleic Acids (LNAs) inwhich the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of thesugar ring thereby forming a bicyclic sugar moiety. The linkage ispreferably a methelyne (—CH₂—)_(n) group bridging the 2′ oxygen atom andthe 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof aredescribed in WO 98/39352 and WO 99/14226.

Other preferred modifications include 2′-methoxy(2′-O—CH₃),2′-aminopropoxy(2′-OCH₂CH₂CH₂ NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, each of which is herein incorporated byreference in its entirety.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C═C—CH₃ or —CH₂—C≡CH) uracil andcytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and3-deazaguanine and 3-deazaadenine. Further modified nucleobases includetricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and thosedisclosed by Englisch et al., Angewandte Chemie, International Edition,1991, 30, 613. Certain of these nucleobases are particularly useful forincreasing the binding affinity of the oligomeric compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by0.6-1.2.degree. C. (Sanghvi et al, Antisense Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications. Representative United Statespatents that teach the preparation of modified nucleobases include, butare not limited to: U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653;5,763,588; 6,005,096; 5,681,941 and 5,750,692, each of which is hereinincorporated by reference.

Another modification of antisense oligonucleotides chemically linking tothe oligonucleotide one or more moieties or conjugates which enhance theactivity, cellular distribution or cellular uptake of theoligonucleotide. The compounds of the invention can include conjugategroups covalently bound to functional groups such as primary orsecondary hydroxyl groups. Conjugate groups of the invention includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugates groups include cholesterols, lipids,cation lipids, phospholipids, cationic phospholipids, biotin, phenazine,folate, phenanthridine, anthraquinone, acridine, fluoresceins,rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamicproperties, in the context of this invention, include groups thatimprove oligomer uptake, enhance oligomer resistance to degradation,and/or strengthen sequence-specific hybridization with RNA. Groups thatenhance the pharmacokinetic properties, in the context of thisinvention, include groups that improve oligomer uptake, distribution,metabolism or excretion. Conjugate moieties include but are not limitedto lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan etal., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20,533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al.,FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of theinvention may also be conjugated to active drug substances, for example,aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen,ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine,2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, abenzothiadiazide, chlorothiazide, a diazepine, indomethicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic. Oligonucleotide-drug conjugates andtheir preparation are described in U.S. patent application Ser. No.09/334,130 (filed Jun. 15, 1999) and U.S. Pat. Nos. 4,828,979;4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538;5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802;5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046;4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941;4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963;5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469;5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241,5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785;5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726;5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is hereinincorporated by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Chimericantisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Preferred chimeric antisense oligonucleotides incorporate at least one2′ modified sugar (preferably 2′-O—(CH₂)₂—O—CH₃) at the 3′ terminal toconfer nuclease resistance and a region with at least 4 contiguous 2′-Hsugars to confer RNase H activity. Such compounds have also beenreferred to in the art as hybrids or gapmers. Preferred gapmers have aregion of 2′ modified sugars (preferably 2′-O—(CH₂)₂—O—CH₃) at the3′-terminal and at the 5′ terminal separated by at least one regionhaving at least 4 contiguous 2′-H sugars and preferably incorporatephosphorothioate backbone linkages. Representative United States patentsthat teach the preparation of such hybrid structures include, but arenot limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007;5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065;5,652,355; 5,652,356; and 5,700,922, each of which is hereinincorporated by reference in its entirety.

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives. The compounds of theinvention may also be admixed, encapsulated, conjugated or otherwiseassociated with other molecules, molecule structures or mixtures ofcompounds, as for example, liposomes, receptor targeted molecules, oral,rectal, topical or other formulations, for assisting in uptake,distribution and/or absorption. Representative United States patentsthat teach the preparation of such uptake, distribution and/orabsorption assisting formulations include, but are not limited to, U.S.Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291;5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899;5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633;5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295;5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756,each of which is herein incorporated by reference.

Other examples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties, suchas those described in WO 90/10048, and other moieties that increasesaffinity of the oligonucleotide for a target nucleic acid sequence, suchas poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. In a preferred procedure, an antisense or sense oligonucleotideis inserted into a suitable retroviral vector. A cell containing thetarget nucleic acid sequence is contacted with the recombinantretroviral vector, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).

Sense or antisense oligonucleotides also may be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target nucleic acid sequence by formation ofan oligonucleotide-lipid complex, as described in WO 90/10448. The senseor antisense oligonucleotide-lipid complex is preferably dissociatedwithin the cell by an endogenous lipase.

Antisense or sense RNA or DNA molecules are generally at least about 5nucleotides in length, alternatively at least about 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110,115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440,450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580,590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720,730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860,870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000nucleotides in length, wherein in this context the term “about” meansthe referenced nucleotide sequence length plus or minus 10% of thatreferenced length.

The probes may also be employed in PCR techniques to generate a pool ofsequences for identification of closely related polypeptide codingsequences.

Nucleotide sequences encoding a polypeptide can also be used toconstruct hybridization probes for mapping the gene which encodes thatpolypeptide and for the genetic analysis of individuals with geneticdisorders. The nucleotide sequences provided herein may be mapped to achromosome and specific regions of a chromosome using known techniques,such as in situ hybridization, linkage analysis against knownchromosomal markers, and hybridization screening with libraries.

The polypeptide can be used in assays to identify other proteins ormolecules involved in a binding interaction with the polypeptide. Bysuch methods, inhibitors of the receptor/ligand binding interaction canbe identified. Proteins involved in such binding interactions can alsobe used to screen for peptide or small molecule inhibitors of thebinding interaction. Screening assays can be designed to find leadcompounds that mimic the biological activity of a native polypeptide ora receptor for the polypeptide. Such screening assays will includeassays amenable to high-throughput screening of chemical libraries,making them particularly suitable for identifying small molecule drugcandidates. Small molecules contemplated include synthetic organic orinorganic compounds. The assays can be performed in a variety offormats, including protein-protein binding assays, biochemical screeningassays, immunoassays and cell based assays, which are well characterizedin the art.

Nucleic acids which encode a polypeptide or its modified forms can alsobe used to generate either transgenic animals or “knock out” animalswhich, in turn, are useful in the development and screening oftherapeutically useful reagents. A transgenic animal (e.g., a mouse orrat) is an animal having cells that contain a transgene, which transgenewas introduced into the animal or an ancestor of the animal at aprenatal, e.g., an embryonic stage. A transgene is a DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops. In one embodiment, cDNA encoding a polypeptide can be used toclone genomic DNA encoding the polypeptide in accordance withestablished techniques and the genomic sequences used to generatetransgenic animals that contain cells which express DNA encoding thepolypeptide. Methods for generating transgenic animals, particularlyanimals such as mice or rats, have become conventional in the art andare described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.Typically, particular cells would be targeted for polypeptide transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding a polypeptide introduced into thegerm line of the animal at an embryonic stage can be used to examine theeffect of increased expression of DNA encoding a polypeptide. Suchanimals can be used as tester animals for reagents thought to conferprotection from, for example, pathological conditions associated withits overexpression. In accordance with this facet of the invention, ananimal is treated with the reagent and a reduced incidence of thepathological condition, compared to untreated animals bearing thetransgene, would indicate a potential therapeutic intervention for thepathological condition.

Alternatively, non-human homologues of a polypeptide can be used toconstruct a gene “knock out” animal which has a defective or alteredgene encoding the polypeptide as a result of homologous recombinationbetween the endogenous gene encoding the polypeptide and altered genomicDNA encoding the polypeptide introduced into an embryonic stem cell ofthe animal. For example, cDNA encoding the polypeptide can be used toclone genomic DNA encoding the polypeptide in accordance withestablished techniques. A portion of the genomic DNA encoding thepolypeptide can be deleted or replaced with another gene, such as a geneencoding a selectable marker which can be used to monitor integration.Typically, several kilobases of unaltered flanking DNA (both at the 5′and 3′ ends) are included in the vector [see e.g., Thomas and Capecchi,Cell, 51:503 (1987) for a description of homologous recombinationvectors]. The vector is introduced into an embryonic stem cell line(e.g., by electroporation) and cells in which the introduced DNA hashomologously recombined with the endogenous DNA are selected [see e.g.,Li et al., Cell, 69:915 (1992)]. The selected cells are then injectedinto a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras [see e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the polypeptide.

Nucleic acid encoding the polypeptides may also be used in gene therapy.In gene therapy applications, genes are introduced into cells in orderto achieve in vivo synthesis of a therapeutically effective geneticproduct, for example for replacement of a defective gene. “Gene therapy”includes both conventional gene therapy where a lasting effect isachieved by a single treatment, and the administration of genetherapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA 83:4143-4146 [1986]). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11, 205-210 [1993]).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and genetherapy protocols see Anderson et al., Science 256, 808-813 (1992).

Methods Involving Screening

The inventions encompass methods of screening compounds to identifythose that prevent the effect of the polypeptide (antagonists) orpromote the effect of the polypeptide (agonist). Screening assays forantagonist drug candidates are designed to identify compounds that bindor complex with the polypeptides encoded by the genes identified herein,or otherwise interfere with the interaction of the encoded polypeptideswith other cellular proteins, including e.g., inhibiting the expressionof the polypeptide from cells. Such screening assays will include assaysamenable to high-throughput screening of chemical libraries, making themparticularly suitable for identifying small molecule drug candidates.

The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

All assays for antagonists are common in that they call for contactingthe drug candidate with a polypeptide encoded by a nucleic acididentified herein under conditions and for a time sufficient to allowthese two components to interact.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. In a particularembodiment, the polypeptide or the drug candidate is immobilized on asolid phase, e.g., on a microtiter plate, by covalent or non-covalentattachments. Non-covalent attachment generally is accomplished bycoating the solid surface with a solution of the polypeptide and drying.Alternatively, an immobilized antibody, e.g., a monoclonal antibody,specific for the polypeptide to be immobilized can be used to anchor itto a solid surface. The assay is performed by adding the non-immobilizedcomponent, which may be labeled by a detectable label, to theimmobilized component, e.g., the coated surface containing the anchoredcomponent. When the reaction is complete, the non-reacted components areremoved, e.g., by washing, and complexes anchored on the solid surfaceare detected. When the originally non-immobilized component carries adetectable label, the detection of label immobilized on the surfaceindicates that complexing occurred. Where the originally non-immobilizedcomponent does not carry a label, complexing can be detected, forexample, by using a labeled antibody specifically binding theimmobilized complex.

If the candidate compound interacts with but does not bind to apolypeptide, its interaction with that polypeptide can be assayed bymethods well known for detecting protein-protein interactions. Suchassays include traditional approaches, such as, e.g., cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers (Fields and Song, Nature (London), 340:245-246 (1989);Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) asdisclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:5789-5793 (1991). Many transcriptional activators, such as yeast GAL4,consist of two physically discrete modular domains, one acting as theDNA-binding domain, the other one functioning as thetranscription-activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the antigen is fused to the DNA-binding domain ofGAL4, and another, in which candidate activating proteins are fused tothe activation domain. The expression of a GAL1-lacZ reporter gene undercontrol of a GAL4-activated promoter depends on reconstitution of GAL4activity via protein-protein interaction. Colonies containinginteracting polypeptides are detected with a chromogenic substrate forβ-galactosidase. A complete kit (MATCHMAKER™) for identifyingprotein-protein interactions between two specific proteins using thetwo-hybrid technique is commercially available from Clontech. Thissystem can also be extended to map protein domains involved in specificprotein interactions as well as to pinpoint amino acid residues that arecrucial for these interactions.

Compounds that interfere with the interaction of a gene encoding apolypeptide identified herein and other intra- or extracellularcomponents can be tested as follows: usually a reaction mixture isprepared containing the product of the gene and the intra- orextracellular component under conditions and for a time allowing for theinteraction and binding of the two products. To test the ability of acandidate compound to inhibit binding, the reaction is run in theabsence and in the presence of the test compound. In addition, a placebomay be added to a third reaction mixture, to serve as positive control.The binding (complex formation) between the test compound and the intra-or extracellular component present in the mixture is monitored asdescribed hereinabove. The formation of a complex in the controlreaction(s) but not in the reaction mixture containing the test compoundindicates that the test compound interferes with the interaction of thetest compound and its reaction partner.

To assay for antagonists, the polypeptide may be added to a cell alongwith the compound to be screened for a particular activity and theability of the compound to inhibit the activity of interest in thepresence of the polypeptide indicates that the compound is an antagonistto the polypeptide. Alternatively, antagonists may be detected bycombining the polypeptide and a potential antagonist with membrane-boundpolypeptide receptors or encoded receptors under appropriate conditionsfor a competitive inhibition assay. The polypeptide can be labeled, suchas by radioactivity, such that the number of polypeptide molecules boundto the receptor can be used to determine the effectiveness of thepotential antagonist. The gene encoding the receptor can be identifiedby numerous methods known to those of skill in the art, for example,ligand panning and FACS sorting. Coligan et al., Current Protocols inImmun., 1(2): Chapter 5 (1991). Preferably, expression cloning isemployed wherein polyadenylated RNA is prepared from a cell responsiveto the polypeptide and a cDNA library created from this RNA is dividedinto pools and used to transfect COS cells or other cells that are notresponsive to the polypeptide. Transfected cells that are grown on glassslides are exposed to labeled polypeptide. The polypeptide can belabeled by a variety of means including iodination or inclusion of arecognition site for a site-specific protein kinase. Following fixationand incubation, the slides are subjected to autoradiographic analysis.Positive pools are identified and sub-pools are prepared andre-transfected using an interactive sub-pooling and re-screeningprocess, eventually yielding a single clone that encodes the putativereceptor.

As an alternative approach for receptor identification, labeledpolypeptide can be photoaffinity-linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE and exposed to X-ray film. The labeled complexcontaining the receptor can be excised, resolved into peptide fragments,and subjected to protein micro-sequencing. The amino acid sequenceobtained from micro-sequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with labeledpolypeptide in the presence of the candidate compound. The ability ofthe compound to enhance or block this interaction could then bemeasured.

More specific examples of potential antagonists include anoligonucleotide that binds to the fusions of immunoglobulin with apolypeptide, and, in particular, antibodies including, withoutlimitation, poly- and monoclonal antibodies and antibody fragments,single-chain antibodies, anti-idiotypic antibodies, and chimeric orhumanized versions of such antibodies or fragments, as well as humanantibodies and antibody fragments. Alternatively, a potential antagonistmay be a closely related protein, for example, a mutated form of thepolypeptide that recognizes the receptor but imparts no effect, therebycompetitively inhibiting the action of the polypeptide.

Another potential antagonist is an antisense RNA or DNA constructprepared using antisense technology, where, e.g., an antisense RNA orDNA molecule acts to block directly the translation of mRNA byhybridizing to targeted mRNA and preventing protein translation.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes themature polypeptides herein, can be used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan etal., Science, 251:1360 (1991)), thereby preventing transcription and theproduction of the polypeptide. The antisense RNA oligonucleotidehybridizes to the mRNA in vivo and blocks translation of the mRNAmolecule into the polypeptide (antisense—Okano, Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression(CRC Press: Boca Raton, Fla., 1988). The oligonucleotides describedabove can also be delivered to cells such that the antisense RNA or DNAmay be expressed in vivo to inhibit production of the polypeptide. Whenantisense DNA is used, oligodeoxyribonucleotides derived from thetranslation-initiation site, e.g., between about −10 and +10 positionsof the target gene nucleotide sequence, are preferred.

Potential antagonists include small molecules that bind to the activesite, the receptor binding site, or growth factor or other relevantbinding site of the polypeptide, thereby blocking the normal biologicalactivity of the polypeptide. Examples of small molecules include, butare not limited to, small peptides or peptide-like molecules, preferablysoluble peptides, and synthetic non-peptidyl organic or inorganiccompounds.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology, 4:469-471 (1994), and PCT publication No. WO 97/33551(published Sep. 18, 1997).

Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT publication No. WO 97/33551, supra.

These small molecules can be identified by any one or more of thescreening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

Isolated polypeptide-encoding nucleic acid can be used for recombinantlyproducing polypeptide using techniques well known in the art and asdescribed herein. In turn, the produced polypeptides can be employed forgenerating antibodies using techniques well known in the art and asdescribed herein.

Antibodies specifically binding a polypeptide identified herein, as wellas other molecules identified by the screening assays disclosedhereinbefore, can be administered for the treatment of variousdisorders, including cancer, in the form of pharmaceutical compositions.

If the polypeptide is intracellular and whole antibodies are used asinhibitors, internalizing antibodies are preferred. However,lipofections or liposomes can also be used to deliver the antibody, oran antibody fragment, into cells. Where antibody fragments are used, thesmallest inhibitory fragment that specifically binds to the bindingdomain of the antigen is preferred. For example, based upon thevariable-region sequences of an antibody, peptide molecules can bedesigned that retain the ability to bind the antigen sequence. Suchpeptides can be synthesized chemically and/or produced by recombinantDNA technology. See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA,90: 7889-7893 (1993).

Pharmaceutical Formulations

Therapeutic formulations comprising an antibody are prepared for storageby mixing the antibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington: The Science and Practice of Pharmacy 20th edition (2000)),in the form of aqueous solutions, lyophilized or other driedformulations. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, histidine and other organicacids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington: The Science and Practice of Pharmacy 20th edition (2000).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the immunoglobulin, which matrices arein the form of shaped articles, e.g., films, or microcapsule. Examplesof sustained-release matrices include polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulatedimmunoglobulins remain in the body for a long time, they may denature oraggregate as a result of exposure to moisture at 37° C., resulting in aloss of biological activity and possible changes in immunogenicity.Rational strategies can be devised for stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

It is further contemplated that an agent useful in the invention can beintroduced to an individual by gene therapy. Gene therapy refers totherapy performed by the administration of a nucleic acid to anindividual. In gene therapy applications, genes are introduced intocells in order to achieve in vivo synthesis of a therapeuticallyeffective genetic product, for example for replacement of a defectivegene. “Gene therapy” includes both conventional gene therapy where alasting effect is achieved by a single treatment, and the administrationof gene therapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. See, e.g., KLβ-SiRNA described inthe Examples. It has already been shown that short antisenseoligonucleotides can be imported into cells where they act asinhibitors, despite their low intracellular concentrations caused bytheir restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA 83:4143-4146 (1986)). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups. For general reviewsof the methods of gene therapy, see, for example, Goldspiel et al.Clinical Pharmacy 12:488-505 (1993); Wu and Wu Biotherapy 3:87-95(1991); Tolstoshev Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan Science 260:926-932 (1993); Morgan and Anderson Ann. Rev.Biochem. 62:191-217 (1993); and May TIBTECH 11:155-215 (1993). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. eds. (1993) Current Protocols inMolecular Biology, John Wiley & Sons, NY; and Kriegler (1990) GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY.

Uses

KLβ modulators may be used in, for example, in vitro, ex vivo and invivo therapeutic methods.

The invention provides methods and compositions useful for modulatingdisease states associated with expression and/or activity of KLβ, suchas increased expression and/or activity or undesired expression and/oractivity, said methods comprising administration of an effective dose ofa KLβ antagonist (such as an anti-KLβ antibody) to an individual in needof such treatment.

In one aspect, the invention provides methods for modulating diseasestates associated with expression and/or activity of KLβ and FGF19, suchas increased expression and/or activity or undesired expression and/oractivity, said methods comprising administration of an effective dose ofa KLβ antagonist (such as an anti-KLβ antibody) to an individual in needof such treatment.

In one aspect, the invention provides methods for modulating diseasestates associated with expression and/or activity of KLβ and FGFR4, suchas increased expression and/or activity or undesired expression and/oractivity, said methods comprising administration of an effective dose ofa KLβ antagonist (such as an anti-KLβ antibody) to an individual in needof such treatment.

One of ordinary skill can determine whether disorders are associatedwith expression and/or activity of KLβ, FGF19 and/or FGFR4 using methodsknown in the art and methods disclosed herein.

It is understood that any suitable KLβ antagonist (such as an anti-KLβantibody) may be used in methods of treatment, including monoclonaland/or polyclonal antibodies, a human antibody, a chimeric antibody, anaffinity-matured antibody, a humanized antibody, and/or an antibodyfragment.

Moreover, at least some of the antibodies can bind antigen from otherspecies. Accordingly, the antibodies can be used to bind specificantigen activity, e.g., in a cell culture containing the antigen, inhuman subjects or in other mammalian subjects having the antigen withwhich an antibody cross-reacts (e.g. chimpanzee, baboon, marmoset,cynomolgus and rhesus, pig or mouse). In one embodiment, the antibodycan be used for inhibiting antigen activities by contacting the antibodywith the antigen such that antigen activity is inhibited. Preferably,the antigen is a human protein molecule.

In one embodiment, an antibody can be used in a method for binding anantigen in an individual suffering from a disorder associated withincreased antigen expression and/or activity, comprising administeringto the subject an antibody such that the antigen in the subject isbound. Preferably, the antigen is a human protein molecule and thesubject is a human subject. Alternatively, the subject can be a mammalexpressing the antigen with which an antibody binds. Still further thesubject can be a mammal into which the antigen has been introduced(e.g., by administration of the antigen or by expression of an antigentransgene). An antibody can be administered to a human subject fortherapeutic purposes. Moreover, an antibody can be administered to anon-human mammal expressing an antigen with which the immunoglobulincross-reacts (e.g., a primate, pig or mouse) for veterinary purposes oras an animal model of human disease. Regarding the latter, such animalmodels may be useful for evaluating the therapeutic efficacy ofantibodies (e.g., testing of dosages and time courses ofadministration).

The antibodies can be used to treat, inhibit, delay progression of,prevent/delay recurrence of, ameliorate, or prevent diseases, disordersor conditions associated with expression and/or activity of one or moreantigen molecules.

In certain embodiments, an immunoconjugate comprising an antibodyconjugated with one or more cytotoxic agent(s) is administered to thepatient. In some embodiments, the immunoconjugate and/or antigen towhich it is bound is/are internalized by the cell, resulting inincreased therapeutic efficacy of the immunoconjugate in killing thetarget cell to which it binds. In one embodiment, the cytotoxic agenttargets or interferes with nucleic acid in the target cell. In oneembodiment, the cytotoxic agent targets or interferes with microtubulepolymerization. Examples of such cytotoxic agents include any of thechemotherapeutic agents noted herein (such as a maytansinoid,auristatin, dolastatin, or a calicheamicin), a radioactive isotope, or aribonuclease or a DNA endonuclease.

In any of the methods herein, one may administer to the subject orpatient along with the KLβ antagonist an effective amount of a secondmedicament (where the antibody herein is a first medicament), which isanother active agent that can treat the condition in the subject thatrequires treatment. For instance, a KLβ antagonist may beco-administered with another KLβ antagonist, an antibody,chemotherapeutic agent(s) (including cocktails of chemotherapeuticagents), anti-angiogenic agent(s), immunosuppressive agents(s),cytokine(s), cytokine antagonist(s), and/or growth-inhibitory agent(s).The type of such second medicament depends on various factors, includingthe type of disorder, such as cancer or an autoimmune disorder, theseverity of the disease, the condition and age of the patient, the typeand dose of first medicament employed, etc.

Where a KLβ antagonist inhibits tumor growth, for example, it may beparticularly desirable to combine it with one or more other therapeuticagents that also inhibit tumor growth. For instance, a KLβ antagonistmay be combined with an anti-angiogenic agent, such as an anti-VEGFantibody (e.g., AVASTIN®) and/or anti-ErbB antibodies (e.g. HERCEPTIN®trastuzumab anti-HER2 antibody or an anti-HER2 antibody that binds toDomain II of HER2, such as OMNITARG™ pertuzumab anti-HER2 antibody) in atreatment scheme, e.g. in treating any of the disease described herein,including hepatocellular carcinoma and pancreatic cancer. Alternatively,or additionally, the patient may receive combined radiation therapy(e.g. external beam irradiation or therapy with a radioactive labeledagent, such as an antibody). Such combined therapies noted above includecombined administration (where the two or more agents are included inthe same or separate formulations), and separate administration, inwhich case, administration of the antibody can occur prior to, and/orfollowing, administration of the adjunct therapy or therapies. Inaddition, combining a KLβ antagonist with a relatively non-cytotoxicagent such as another biologic molecule, e.g., an antibody is expectedto reduce cytotoxicity versus combining the KLβ antagonist with achemotherapeutic agent of other agent that is highly toxic to cells.

Treatment with a combination of a KLβ antagonist with one or more secondmedicaments preferably results in an improvement in the signs orsymptoms of cancer. For instance, such therapy may result in animprovement in survival (overall survival and/or progression-freesurvival) relative to a patient treated with the second medicament only(e.g., a chemotherapeutic agent only), and/or may result in an objectiveresponse *(partial or complete, preferably complete). Moreover,treatment with the combination of a KLβ antagonist and one or moresecond medicament(s) preferably results in an additive, and morepreferably synergistic (or greater than additive), therapeutic benefitto the patient. Preferably, in this combination method the timingbetween at least one administration of the second medicament and atleast one administration of the KLβ antagonist is about one month orless, more preferably, about two weeks or less.

For treatment of cancers, the second medicament is preferably anotherKLβ antagonist, an antibody, chemotherapeutic agent (including cocktailsof chemotherapeutic agents), anti-angiogenic agent, immunosuppressiveagent, prodrug, cytokine, cytokine antagonist, cytotoxic radiotherapy,corticosteroid, anti-emetic, cancer vaccine, analgesic, anti-vascularagent, and/or growth-inhibitory agent. The cytotoxic agent includes anagent interacting with DNA, the antimetabolites, the topoisomerase I orII inhibitors, or the spindle inhibitor or stabilizer agents (e.g.,preferably vinca alkaloid, more preferably selected from vinblastine,deoxyvinblastine, vincristine, vindesine, vinorelbine, vinepidine,vinfosiltine, vinzolidine and vinfunine), or any agent used inchemotherapy such as 5-FU, a taxane, doxorubicin, or dexamethasone.

In another embodiment, the second medicament is an antibody used totreat cancers such as those directed against the extracellular domain ofthe HER2/neu receptor, e.g., trastuzumab, or one of its functionalfragments, pan-HER inhibitor, a Src inhibitor, a MEK inhibitor, or anEGFR inhibitor (e.g., an anti-EGFR antibody (such as one inhibiting thetyrosine kinase activity of the EGFR), which is preferably the mousemonoclonal antibody 225, its mouse-man chimeric derivative C225, or ahumanized antibody derived from this antibody 225 or derived naturalagents, dianilinophthalimides, pyrazolo- or pyrrolopyridopyrimidines,quinazilines, gefitinib, erlotinib, cetuximab, ABX-EFG, canertinib,EKB-569 and PKI-166), or dual-EGFR/HER-2 inhibitor such as lapatanib.Additional second medicaments include alemtuzumab (CAMPATH™), FavID(IDKLH), CD20 antibodies with altered glycosylation, such asGA-101/GLYCART™, oblimersen (GENASENSE™), thalidomide and analogsthereof, such as lenalidomide (REVLIMID™), imatinib, sorafenib,ofatumumab (HUMAX-CD20™), anti-CD40 antibody, e.g. SGN-40, andanti-CD-80 antibody, e.g. galiximab.

The anti-emetic agent is preferably ondansetron hydrochloride,granisetron hydrochloride, metroclopramide, domperidone, haloperidol,cyclizine, lorazepam, prochlorperazine, dexamethasone, levomepromazine,or tropisetron. The vaccine is preferably GM-CSF DNA and cell-basedvaccines, dendritic cell vaccine, recombinant viral vaccines, heat shockprotein (HSP) vaccines, allogeneic or autologous tumor vaccines. Theanalgesic agent preferably is ibuprofen, naproxen, choline magnesiumtrisalicylate, or oxycodone hydrochloride. The anti-vascular agentpreferably is bevacizumab, or rhuMAb-VEGF. Further second medicamentsinclude anti-proliferative agents such a farnesyl protein transferaseinhibitors, anti-VEGF inhibitors, p53 inhibitors, or PDGFR inhibitors.The second medicament herein includes also biologic-targeted therapysuch as treatment with antibodies as well as small-molecule-targetedtherapy, for example, against certain receptors.

Many anti-angiogenic agents have been identified and are known in theart, including those listed herein, e.g., listed under Definitions, andby, e.g., Carmeliet and Jain, Nature 407:249-257 (2000); Ferrara et al.,Nature Reviews:Drug Discovery, 3:391-400 (2004); and Sato Int. J. Clin.Oncol., 8:200-206 (2003). See also, US Patent Application US20030055006.In one embodiment, an anti-KLβ antibody is used in combination with ananti-VEGF neutralizing antibody (or fragment) and/or another VEGFantagonist or a VEGF receptor antagonist including, but not limited to,for example, soluble VEGF receptor (e.g., VEGFR-1, VEGFR-2, VEGFR-3,neuropillins (e.g., NRP1, NRP2)) fragments, aptamers capable of blockingVEGF or VEGFR, neutralizing anti-VEGFR antibodies, low molecule weightinhibitors of VEGFR tyrosine kinases (RTK), antisense strategies forVEGF, ribozymes against VEGF or VEGF receptors, antagonist variants ofVEGF; and any combinations thereof. Alternatively, or additionally, twoor more angiogenesis inhibitors may optionally be co-administered to thepatient in addition to VEGF antagonist and other agent. In certainembodiment, one or more additional therapeutic agents, e.g., anti-canceragents, can be administered in combination with a KLβ antagonist (suchas an anti-KLβ antibody), the VEGF antagonist, and an anti-angiogenesisagent.

Chemotherapeutic agents useful herein are described supra, e.g., in thedefinition of “chemotherapeutic agent”.

Exemplary second medicaments include an alkylating agent, a folateantagonist, a pyrimidine antagonist, a cytotoxic antibiotic, a platinumcompound or platinum-based compound, a taxane, a vinca alkaloid, a c-Kitinhibitor, a topoisomerase inhibitor, an anti-angiogenesis inhibitorsuch as an anti-VEGF inhibitor, a HER-2 inhibitor, an EGFR inhibitor ordual EGFR/HER-2 kinase inhibitor, an anti-estrogen such as fulvestrant,and a hormonal therapy agent, such as carboplatin, cisplatin,gemcitabine, capecitabine, epirubicin, tamoxifen, an aromataseinhibitor, and prednisone. Most preferably, the cancer is colorectalcancer and the second medicament is an EGFR inhibitor such as erlotinib,an anti-VEGF inhibitor such as bevacizumab, or is cetuximab, arinotecan,irinotecan, or FOLFOX, or the cancer is breast cancer an the secondmedicament is an anti-estrogen modulator such as fulvestrant, tamoxifenor an aromatase inhibitor such as letrozole, exemestane, or anastrozole,or is a VEGF inhibitor such as bevacizumab, or is a chemotherapeuticagent such as doxorubicin, and/or a taxane such as paclitaxel, or is ananti-HER-2 inhibitor such as trastuzumab, or a dual EGFR/HER-2 kinaseinhibitor such as lapatinib or a HER-2 downregulator such as 17AAG(geldanamycin derivative that is a heat shock protein [Hsp] 90 poison)(for example, for breast cancers that have progressed on trastuzumab).In other embodiments, the cancer is lung cancer, such as small-cell lungcancer, and the second medicament is a VEGF inhibitor such asbevacizumab, or an EGFR inhibitor such as, e.g., erlotinib or a c-Kitinhibitor such as e.g., imatinib. In other embodiments, the cancer isliver cancer, such as hepatocellular carcinoma, and the secondmedicament is an EGFR inhibitor such as erlotinib, a chemotherapeuticagent such as doxorubicin or irinotecan, a taxane such as paclitaxel,thalidomide and/or interferon. Further, a preferred chemotherapeuticagent for front-line therapy of cancer is taxotere, alone in combinationwith other second medicaments. Most preferably, if chemotherapy isadministered, it is given first, followed by the antibodies.

Such second medicaments may be administered within 48 hours after theantibodies are administered, or within 24 hours, or within 12 hours, orwithin 3-12 hours after said agent, or may be administered over apre-selected period of time, which is preferably about 1 to 2 days.Further, the dose of such agent may be sub-therapeutic.

The KLβ antagonist can be administered concurrently, sequentially, oralternating with the second medicament or upon non-responsiveness withother therapy. Thus, the combined administration of a second medicamentincludes co-administration (concurrent administration), using separateformulations or a single pharmaceutical formulation, and consecutiveadministration in either order, wherein preferably there is a timeperiod while both (or all) medicaments simultaneously exert theirbiological activities. All these second medicaments may be used incombination with each other or by themselves with the first medicament,so that the express “second medicament” as used herein does not mean itis the only medicament besides the first medicament, respectively. Thus,the second medicament need not be one medicament, but may constitute orcomprise more than one such drug.

These second medicaments as set forth herein may be used in the samedosages and with administration routes as the first medicaments, orabout from 1 to 99% of the dosages of the first medicaments. If suchsecond medicaments are used at all, preferably, they are used in loweramounts than if the first medicament were not present, especially insubsequent dosings beyond the initial dosing with the first medicament,so as to eliminate or reduce side effects caused thereby.

The invention also provides methods and compositions for inhibiting orpreventing relapse tumor growth or relapse cancer cell growth. Relapsetumor growth or relapse cancer cell growth is used to describe acondition in which patients undergoing or treated with one or morecurrently available therapies (e.g., cancer therapies, such aschemotherapy, radiation therapy, surgery, hormonal therapy and/orbiological therapy/immunotherapy, anti-VEGF antibody therapy,particularly a standard therapeutic regimen for the particular cancer)is not clinically adequate to treat the patients or the patients are nolonger receiving any beneficial effect from the therapy such that thesepatients need additional effective therapy. As used herein, the phrasecan also refer to a condition of the “non-responsive/refractory”patient, e.g., which describe patients who respond to therapy yet sufferfrom side effects, develop resistance, do not respond to the therapy, donot respond satisfactorily to the therapy, etc. In various embodiments,a cancer is relapse tumor growth or relapse cancer cell growth where thenumber of cancer cells has not been significantly reduced, or hasincreased, or tumor size has not been significantly reduced, or hasincreased, or fails any further reduction in size or in number of cancercells. The determination of whether the cancer cells are relapse tumorgrowth or relapse cancer cell growth can be made either in vivo or invitro by any method known in the art for assaying the effectiveness oftreatment on cancer cells, using the art-accepted meanings of “relapse”or “refractory” or “non-responsive” in such a context. A tumor resistantto anti-VEGF treatment is an example of a relapse tumor growth.

The invention provides methods of blocking or reducing relapse tumorgrowth or relapse cancer cell growth in an individual by administeringone or more KLβ antagonist (such as an anti-KLβ antibody) to block orreduce the relapse tumor growth or relapse cancer cell growth insubject. In certain embodiments, the KLβ antagonist can be administeredsubsequent to the cancer therapeutic. In certain embodiments, the KLβantagonist is administered simultaneously with cancer therapy.Alternatively, or additionally, the KLβ antagonist therapy alternateswith another cancer therapy, which can be performed in any order. Theinvention also encompasses methods for administering one or moreinhibitory antibodies to prevent the onset or recurrence of cancer inpatients predisposed to having cancer. Generally, the subject was or isconcurrently undergoing cancer therapy. In one embodiment, the cancertherapy is treatment with an anti-angiogenesis agent, e.g., a VEGFantagonist. The anti-angiogenesis agent includes those known in the artand those found under the Definitions herein. In one embodiment, theanti-angiogenesis agent is an anti-VEGF neutralizing antibody orfragment (e.g., humanized A4.6.1, AVASTIN® (Genentech, South SanFrancisco, Calif.), Y0317, M4, G6, B20, 2C3, etc.). See, e.g., U.S. Pat.Nos. 6,582,959, 6,884,879, 6,703,020; WO98/45332; WO 96/30046;WO94/10202; EP 0666868B1; US Patent Applications 20030206899,20030190317, 20030203409, and 20050112126; Popkov et al., Journal ofImmunological Methods 288:149-164 (2004); and, WO2005012359. Additionalagents can be administered in combination with VEGF antagonist and a KLβantagonist for blocking or reducing relapse tumor growth or relapsecancer cell growth.

The KLβ antagonists (and adjunct therapeutic agent) is/are administeredby any suitable means, including parenteral, subcutaneous,intraperitoneal, intrapulmonary, and intranasal, and, if desired forlocal treatment, intralesional administration. Parenteral infusionsinclude intramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the KLβ antagonists aresuitably administered by pulse infusion, particularly with decliningdoses of the antibody. Dosing can be by any suitable route, e.g. byinjections, such as intravenous or subcutaneous injections, depending inpart on whether the administration is brief or chronic.

The KLβ antagonist composition will be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. TheKLβ antagonist need not be, but is optionally formulated with one ormore agents currently used to prevent or treat the disorder in question.The effective amount of such other agents depends on the amount of KLβantagonist present in the formulation, the type of disorder ortreatment, and other factors discussed above. These are generally usedin the same dosages and with administration routes as used hereinbeforeor about from 1 to 99% of the heretofore employed dosages.

For the prevention or treatment of disease, the appropriate dosage of aKLβ antagonist (when used alone or in combination with other agents)will depend on the type of disease to be treated, the type of antibody,the severity and course of the disease, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The antibody is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of an anti-KLβ antibody is an initialcandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. One typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. One exemplary dosage of the antibody would be in therange from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more dosesof about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combinationthereof) may be administered to the patient. Such doses may beadministered intermittently, e.g. every week or every three weeks (e.g.such that the patient receives from about two to about twenty, e.g.about six doses of the antibody). An initial higher loading dose,followed by one or more lower doses may be administered. An exemplarydosing regimen comprises administering an initial loading dose of about4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of theantibody. However, other dosage regimens may be useful. The progress ofthis therapy is easily monitored by conventional techniques and assays.

Uses Comprising Detection of KLβ

In another aspect, the invention provides methods for detection of KLβ,the methods comprising detecting KLβ in a sample. The term “detection”as used herein includes qualitative and/or quantitative detection(measuring levels) with or without reference to a control.

In one aspect, the invention provides methods for detecting a disorderassociated with KLβ expression and/or activity, the methods comprisingdetecting KLβ in a biological sample from an individual. In someembodiments, the KLβ expression is increased expression or abnormalexpression. In some embodiments, the disorder is a tumor, cancer, and/ora cell proliferative disorder (such as hepatocellular carcinoma andpancreatic cancer), a liver disorder (such as cirrhosis), or anydisorder described herein. In some embodiment, the biological sample isserum or of a tumor.

For example, a sample may be assayed for a target antigen (e.g., KLβ,FGF19, and/or FGFR4) by obtaining the sample from a desired source,admixing the sample with anti-target antigen antibody to allow theantibody to form antibody/target antigen complex with any present in themixture, and detecting any antibody/target antigen complex present inthe mixture. The biological sample may be prepared for assay by methodsknown in the art which are suitable for the particular sample. Themethods of admixing the sample with antibodies and the methods ofdetecting antibody/target antigen complex are chosen according to thetype of assay used. Such assays include immunohistochemistry,competitive and sandwich assays, and steric inhibition assays. Forsample preparation, a tissue or cell sample from a mammal (typically ahuman patient) may be used. Examples of samples include, but are notlimited to, cancer cells such as colon, breast, prostate, ovary, lung,stomach, pancreas, lymphoma, and leukemia cancer cells. Target antigenmay also be measured in serum. The sample can be obtained by a varietyof procedures known in the art including, but not limited to surgicalexcision, aspiration or biopsy. The tissue may be fresh or frozen. Inone embodiment, the sample is fixed and embedded in paraffin or thelike. The tissue sample may be fixed (i.e. preserved) by conventionalmethodology (See e.g., “Manual of Histological Staining Method of theArmed Forces Institute of Pathology,” 3^(rd) edition (1960) Lee G. Luna,H T (ASCP) Editor, The Blakston Division McGraw-Hill Book Company, NewYork; The Armed Forces Institute of Pathology Advanced LaboratoryMethods in Histology and Pathology (1994) Ulreka V. Mikel, Editor, ArmedForces Institute of Pathology, American Registry of Pathology,Washington, D.C.). One of ordinary skill in the art will appreciate thatthe choice of a fixative is determined by the purpose for which thesample is to be histologically stained or otherwise analyzed. One ofordinary skill in the art will also appreciate that the length offixation depends upon the size of the tissue sample and the fixativeused. By way of example, neutral buffered formalin, Bouin's orparaformaldehyde, may be used to fix a sample. Generally, the sample isfirst fixed and is then dehydrated through an ascending series ofalcohols, infiltrated and embedded with paraffin or other sectioningmedia so that the tissue sample may be sectioned. Alternatively, one maysection the tissue and fix the sections obtained. By way of example, thetissue sample may be embedded and processed in paraffin by conventionalmethodology (See e.g., “Manual of Histological Staining Method of theArmed Forces Institute of Pathology”, supra). Examples of paraffin thatmay be used include, but are not limited to, Paraplast, Broloid, andTissuemay. Once the tissue sample is embedded, the sample may besectioned by a microtome or the like (See e.g., “Manual of HistologicalStaining Method of the Armed Forces Institute of Pathology”, supra). Byway of example for this procedure, sections may range from about threemicrons to about five microns in thickness. Once sectioned, the sectionsmay be attached to slides by several standard methods. Examples of slideadhesives include, but are not limited to, silane, gelatin,poly-L-lysine and the like. By way of example, the paraffin embeddedsections may be attached to positively charged slides and/or slidescoated with poly-L-lysine. If paraffin has been used as the embeddingmaterial, the tissue sections are generally deparaffinized andrehydrated to water. The tissue sections may be deparaffinized byseveral conventional standard methodologies. For example, xylenes and agradually descending series of alcohols may be used (See e.g., “Manualof Histological Staining Method of the Armed Forces Institute ofPathology”, supra). Alternatively, commercially availabledeparaffinizing non-organic agents such as Hemo-De7 (CMS, Houston, Tex.)may be used.

Anti-KLβ antibodies are useful in assays detecting KLβ expression (suchas diagnostic or prognostic assays) in specific cells or tissues whereinthe antibodies are labeled as described below and/or are immobilized onan insoluble matrix. However, it is understood that any suitableanti-KLβ antibody may be used in embodiments involving detection anddiagnosis. Some methods for making anti-KLβ antibodies are describedherein and methods for making anti-KLβ antibodies are well known in theart, e.g., antibodies disclosed in Ito et al (2005) J Clin Invest115(8): 2202-2208; R&D Systems Catalog Nos. MAB3738 and AF2619.

Analytical methods a for target antigen all use one or more of thefollowing reagents: labeled target antigen analogue, immobilized targetantigen analogue, labeled anti-target antigen antibody, immobilizedanti-target antigen antibody and steric conjugates. The labeled reagentsalso are known as “tracers.”

The label used is any detectable functionality that does not interferewith the binding of target antigen and anti-target antigen antibody.Numerous labels are known for use in immunoassay, examples includingmoieties that may be detected directly, such as fluorochrome,chemiluminescent, and radioactive labels, as well as moieties, such asenzymes, that must be reacted or derivatized to be detected.

The label used is any detectable functionality that does not interferewith the binding of target antigen and anti-target antigen antibody.Numerous labels are known for use in immunoassay, examples includingmoieties that may be detected directly, such as fluorochrome,chemiluminescent, and radioactive labels, as well as moieties, such asenzymes, that must be reacted or derivatized to be detected. Examples ofsuch labels include the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

Conventional methods are available to bind these labels covalently toproteins or polypeptides. For instance, coupling agents such asdialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotizedbenzidine, and the like may be used to tag the antibodies with theabove-described fluorescent, chemiluminescent, and enzyme labels. See,for example, U.S. Pat. No. 3,940,475 (fluorimetry) and 3,645,090(enzymes); Hunter et al., Nature, 144: 945 (1962); David et al.,Biochemistry, 13: 1014-1021 (1974); Pain et al., J. Immunol. Methods,40: 219-230 (1981); and Nygren, J. Histochem. and Cytochem., 30: 407-412(1982). Preferred labels herein are enzymes such as horseradishperoxidase and alkaline phosphatase. The conjugation of such label,including the enzymes, to the antibody is a standard manipulativeprocedure for one of ordinary skill in immunoassay techniques. See, forexample, O'Sullivan et al., “Methods for the Preparation ofEnzyme-antibody Conjugates for Use in Enzyme Immunoassay,” in Methods inEnzymology, ed. J. J. Langone and H. Van Vunakis, Vol. 73 (AcademicPress, New York, N.Y., 1981), pp. 147-166.

Immobilization of reagents is required for certain assay methods.Immobilization entails separating the anti-target antigen antibody fromany target antigen that remains free in solution. This conventionally isaccomplished by either insolubilizing the anti-target antigen antibodyor target antigen analogue before the assay procedure, as by adsorptionto a water-insoluble matrix or surface (Bennich et al., U.S. Pat. No.3,720,760), by covalent coupling (for example, using glutaraldehydecross-linking), or by insolubilizing the anti-target antigen antibody ortarget antigen analogue afterward, e.g., by immunoprecipitation.

The expression of proteins in a sample may be examined usingimmunohistochemistry and staining protocols. Immunohistochemicalstaining of tissue sections has been shown to be a reliable method ofassessing or detecting presence of proteins in a sample.Immunohistochemistry (“IHC”) techniques utilize an antibody to probe andvisualize cellular antigens in situ, generally by chromogenic orfluorescent methods. For sample preparation, a tissue or cell samplefrom a mammal (typically a human patient) may be used. The sample can beobtained by a variety of procedures known in the art including, but notlimited to surgical excision, aspiration or biopsy. The tissue may befresh or frozen. In one embodiment, the sample is fixed and embedded inparaffin or the like. The tissue sample may be fixed (i.e. preserved) byconventional methodology. One of ordinary skill in the art willappreciate that the choice of a fixative is determined by the purposefor which the sample is to be histologically stained or otherwiseanalyzed. One of ordinary skill in the art will also appreciate that thelength of fixation depends upon the size of the tissue sample and thefixative used.

IHC may be performed in combination with additional techniques such asmorphological staining and/or fluorescence in-situ hybridization. Twogeneral methods of IHC are available; direct and indirect assays.According to the first assay, binding of antibody to the target antigen(e.g., KLβ) is determined directly. This direct assay uses a labeledreagent, such as a fluorescent tag or an enzyme-labeled primaryantibody, which can be visualized without further antibody interaction.In a typical indirect assay, unconjugated primary antibody binds to theantigen and then a labeled secondary antibody binds to the primaryantibody. Where the secondary antibody is conjugated to an enzymaticlabel, a chromogenic or fluorogenic substrate is added to providevisualization of the antigen. Signal amplification occurs becauseseveral secondary antibodies may react with different epitopes on theprimary antibody.

The primary and/or secondary antibody used for immunohistochemistrytypically will be labeled with a detectable moiety. Numerous labels areavailable which can be generally grouped into the following categories:

Aside from the sample preparation procedures discussed above, furthertreatment of the tissue section prior to, during or following IHC may bedesired, For example, epitope retrieval methods, such as heating thetissue sample in citrate buffer may be carried out (see, e.g., Leong etal. Appl. Immunohistochem. 4(3):201 (1996)).

Following an optional blocking step, the tissue section is exposed toprimary antibody for a sufficient period of time and under suitableconditions such that the primary antibody binds to the antigen in thetissue sample. Appropriate conditions for achieving this can bedetermined by routine experimentation. The extent of binding of antibodyto the sample is determined by using any one of the detectable labelsdiscussed above. Preferably, the label is an enzymatic label (e.g. HRPO)which catalyzes a chemical alteration of the chromogenic substrate suchas 3,3′-diaminobenzidine chromogen. Preferably the enzymatic label isconjugated to antibody which binds specifically to the primary antibody(e.g. the primary antibody is rabbit polyclonal antibody and secondaryantibody is goat anti-rabbit antibody).

Specimens thus prepared may be mounted and coverslipped. Slideevaluation is then determined, e.g. using a microscope, and stainingintensity criteria, routinely used in the art, may be employed.

Other assay methods, known as competitive or sandwich assays, are wellestablished and widely used in the commercial diagnostics industry.

Competitive assays rely on the ability of a tracer target antigenanalogue to compete with the test sample target antigen for a limitednumber of anti-target antigen antibody antigen-binding sites. Theanti-target antigen antibody generally is insolubilized before or afterthe competition and then the tracer and target antigen bound to theanti-target antigen antibody are separated from the unbound tracer andtarget antigen. This separation is accomplished by decanting (where thebinding partner was preinsolubilized) or by centrifuging (where thebinding partner was precipitated after the competitive reaction). Theamount of test sample target antigen is inversely proportional to theamount of bound tracer as measured by the amount of marker substance.Dose-response curves with known amounts of target antigen are preparedand compared with the test results to quantitatively determine theamount of target antigen present in the test sample. These assays arecalled ELISA systems when enzymes are used as the detectable markers.

Another species of competitive assay, called a “homogeneous” assay, doesnot require a phase separation. Here, a conjugate of an enzyme with thetarget antigen is prepared and used such that when anti-target antigenantibody binds to the target antigen the presence of the anti-targetantigen antibody modifies the enzyme activity. In this case, the targetantigen or its immunologically active fragments are conjugated with abifunctional organic bridge to an enzyme such as peroxidase. Conjugatesare selected for use with anti-target antigen antibody so that bindingof the anti-target antigen antibody inhibits or potentiates the enzymeactivity of the label. This method per se is widely practiced under thename of EMIT.

Steric conjugates are used in steric hindrance methods for homogeneousassay. These conjugates are synthesized by covalently linking alow-molecular-weight hapten to a small target antigen fragment so thatantibody to hapten is substantially unable to bind the conjugate at thesame time as anti-target antigen antibody. Under this assay procedurethe target antigen present in the test sample will bind anti-targetantigen antibody, thereby allowing anti-hapten to bind the conjugate,resulting in a change in the character of the conjugate hapten, e.g., achange in fluorescence when the hapten is a fluorophore.

Sandwich assays particularly are useful for the determination of targetantigen or anti-target antigen antibodies. In sequential sandwich assaysan immobilized anti-target antigen antibody is used to adsorb testsample target antigen, the test sample is removed as by washing, thebound target antigen is used to adsorb a second, labeled anti-targetantigen antibody and bound material is then separated from residualtracer. The amount of bound tracer is directly proportional to testsample target antigen. In “simultaneous” sandwich assays the test sampleis not separated before adding the labeled anti-target antigen. Asequential sandwich assay using an anti-target antigen monoclonalantibody as one antibody and a polyclonal anti-target antigen antibodyas the other is useful in testing samples for target antigen.

The foregoing are merely exemplary detection assays for target antigen.Other methods now or hereafter developed that use anti-target antigenantibody for the determination of target antigen are included within thescope hereof, including the bioassays described herein.

In one aspect, the invention provides methods to detect (e.g., presenceor absence of or amount) a polynucleotide(s) (e.g., targetpolynucleotides) in a biological sample from an individual, such as ahuman subject. A variety of methods for detecting polynucleotides can beemployed and include, for example, RT-PCR, taqman, amplificationmethods, polynucleotide microarray, and the like.

Methods for the detection of polynucleotides (such as mRNA) are wellknown and include, for example, hybridization assays using complementaryDNA probes (such as in situ hybridization using labeled targetriboprobes), Northern blot and related techniques, and various nucleicacid amplification assays (such as RT-PCR using complementary primersspecific for target, and other amplification type detection methods,such as, for example, branched DNA, SPIA, Ribo-SPLA, SISBA, TMA and thelike).

Biological samples from mammals can be conveniently assayed for, e.g.,target mRNAs using Northern, dot blot or PCR analysis. For example,RT-PCR assays such as quantitative PCR assays are well known in the art.In an illustrative embodiment, a method for detecting target mRNA in abiological sample comprises producing cDNA from the sample by reversetranscription using at least one primer; amplifying the cDNA so producedusing a target polynucleotide as sense and antisense primers to amplifytarget cDNAs therein; and detecting the presence or absence of theamplified target cDNA. In addition, such methods can include one or moresteps that allow one to determine the amount (levels) of target mRNA ina biological sample (e.g. by simultaneously examining the levels acomparative control mRNA sequence of a housekeeping gene such as anactin family member). Optionally, the sequence of the amplified targetcDNA can be determined.

Probes and/or primers may be labeled with a detectable marker, such as,for example, a radioisotope, fluorescent compound, bioluminescentcompound, a chemiluminescent compound, metal chelator or enzyme. Suchprobes and primers can be used to detect the presence of targetpolynucleotides in a sample and as a means for detecting a cellexpressing antigens. As will be understood by the skilled artisan, agreat many different primers and probes may be prepared (e.g., based onthe sequences provided in herein) and used effectively to amplify, cloneand/or determine the presence or absence of and/or amount of targetmRNAs.

Optional methods of the invention include protocols comprising detectionof polynucleotides, such as target polynucleotide, in a tissue or cellsample using microarray technologies. For example, using nucleic acidmicroarrays, test and control mRNA samples from test and control tissuesamples are reverse transcribed and labeled to generate cDNA probes. Theprobes are then hybridized to an array of nucleic acids immobilized on asolid support. The array is configured such that the sequence andposition of each member of the array is known. For example, a selectionof genes that have potential to be expressed in certain disease statesmay be arrayed on a solid support. Hybridization of a labeled probe witha particular array member indicates that the sample from which the probewas derived expresses that gene. Differential gene expression analysisof disease tissue can provide valuable information. Microarraytechnology utilizes nucleic acid hybridization techniques and computingtechnology to evaluate the mRNA expression profile of thousands of geneswithin a single experiment. (see, e.g., WO 01/75166 published Oct. 11,2001; (See, for example, U.S. Pat. No. 5,700,637, U.S. Pat. No.5,445,934, and U.S. Pat. No. 5,807,522, Lockart, Nature Biotechnology,14:1675-1680 (1996); Cheung, V. G. et al., Nature Genetics21(Suppl):15-19 (1999) for a discussion of array fabrication). DNAmicroarrays are miniature arrays containing gene fragments that areeither synthesized directly onto or spotted onto glass or othersubstrates. Thousands of genes are usually represented in a singlearray. A typical microarray experiment involves the following steps: 1.preparation of fluorescently labeled target from RNA isolated from thesample, 2. hybridization of the labeled target to the microarray, 3.washing, staining, and scanning of the array, 4. analysis of the scannedimage and 5. generation of gene expression profiles. Currently two maintypes of DNA microarrays are being used: oligonucleotide (usually 25 to70 mers) arrays and gene expression arrays containing PCR productsprepared from cDNAs. In forming an array, oligonucleotides can be eitherprefabricated and spotted to the surface or directly synthesized on tothe surface (in situ).

The Affymetrix GeneChip® system is a commercially available microarraysystem which comprises arrays fabricated by direct synthesis ofoligonucleotides on a glass surface. Probe/Gene Arrays:Oligonucleotides, usually 25 mers, are directly synthesized onto a glasswafer by a combination of semiconductor-based photolithography and solidphase chemical synthesis technologies. Each array contains up to 400,000different oligos and each oligo is present in millions of copies. Sinceoligonucleotide probes are synthesized in known locations on the array,the hybridization patterns and signal intensities can be interpreted interms of gene identity and relative expression levels by the AffymetrixMicroarray Suite software. Each gene is represented on the array by aseries of different oligonucleotide probes. Each probe pair consists ofa perfect match oligonucleotide and a mismatch oligonucleotide. Theperfect match probe has a sequence exactly complimentary to theparticular gene and thus measures the expression of the gene. Themismatch probe differs from the perfect match probe by a single basesubstitution at the center base position, disturbing the binding of thetarget gene transcript. This helps to determine the background andnonspecific hybridization that contributes to the signal measured forthe perfect match oligo. The Microarray Suite software subtracts thehybridization intensities of the mismatch probes from those of theperfect match probes to determine the absolute or specific intensityvalue for each probe set. Probes are chosen based on current informationfrom GenBank and other nucleotide repositories. The sequences arebelieved to recognize unique regions of the 3′ end of the gene. AGeneChip Hybridization Oven (“rotisserie” oven) is used to carry out thehybridization of up to 64 arrays at one time. The fluidics stationperforms washing and staining of the probe arrays. It is completelyautomated and contains four modules, with each module holding one probearray. Each module is controlled independently through Microarray Suitesoftware using preprogrammed fluidics protocols. The scanner is aconfocal laser fluorescence scanner which measures fluorescenceintensity emitted by the labeled cRNA bound to the probe arrays. Thecomputer workstation with Microarray Suite software controls thefluidics station and the scanner. Microarray Suite software can controlup to eight fluidics stations using preprogrammed hybridization, wash,and stain protocols for the probe array. The software also acquires andconverts hybridization intensity data into a presence/absence call foreach gene using appropriate algorithms. Finally, the software detectschanges in gene expression between experiments by comparison analysisand formats the output into .txt files, which can be used with othersoftware programs for further data analysis.

In some embodiments, gene deletion, gene mutation, or gene amplificationis detected (eg, KLβ and/or FGFR4 and/or FGF19 gene deletion, genemutation, or gene amplification). Gene deletion, gene mutation, oramplification may be measured by any one of a wide variety of protocolsknown in the art, for example, by conventional Southern blotting,Northern blotting to quantitate the transcription of mRNA (Thomas, Proc.Natl. Acad. Sci. USA, 77:5201-5205 (1980)), dot blotting (DNA analysis),or in situ hybridization (e.g., FISH), using an appropriately labeledprobe, cytogenetic methods or comparative genomic hybridization (CGH)using an appropriately labeled probe. In addition, these methods may beemployed to detect target gene deletion, ligand mutation, or geneamplification. As used herein, “detecting KLβ expression” encompassesdetection of KLβ gene deletion, gene mutation or gene amplification.

Additionally, one can examine the methylation status of the target genein a tissue or cell sample. Aberrant demethylation and/orhypermethylation of CpG islands in gene 5′ regulatory regions frequentlyoccurs in immortalized and transformed cells, and can result in alteredexpression of various genes. A variety of assays for examiningmethylation status of a gene are well known in the art. For example, onecan utilize, in Southern hybridization approaches, methylation-sensitiverestriction enzymes which cannot cleave sequences that containmethylated CpG sites to assess the methylation status of CpG islands. Inaddition, MSP (methylation specific PCR) can rapidly profile themethylation status of all the CpG sites present in a CpG island of agiven gene. This procedure involves initial modification of DNA bysodium bisulfite (which will convert all unmethylated cytosines touracil) followed by amplification using primers specific for methylatedversus unmethylated DNA. Protocols involving methylation interferencecan also be found for example in Current Protocols In Molecular Biology,Unit 12, Frederick M. Ausubel et al. eds., 1995; De Marzo et al., Am. J.Pathol. 155(6): 1985-1992 (1999); Brooks et al, Cancer Epidemiol.Biomarkers Prey., 1998, 7:531-536); and Lethe et al., Int. J. Cancer76(6): 903-908 (1998). As used herein, “detecting KLβ expression”encompasses detection of KLβ gene methylation.

In some embodiments, using methods known in the art, including thosedescribed herein, the polynucleotide and/or polypeptide expression ofone or more targets can be detected. By way of example, the IHCtechniques described above may be employed to detect the presence of oneor more such molecules in the sample. As used herein, “in conjunction”is meant to encompass any simultaneous and/or sequential detection.Thus, it is contemplated that in embodiments in which a biologicalsample is being examined not only for the presence of a first target,but also for the presence of Fa second target, separate slides may beprepared from the same tissue or sample, and each slide tested with areagent that binds to the first and/or second target, respectively.Alternatively, a single slide may be prepared from the tissue or cellsample, and antibodies directed to the first and second target,respectively, may be used in connection with a multi-color stainingprotocol to allow visualization and detection of the first and secondtarget.

Biological samples are described herein, e.g., in the definition ofBiological Sample. In some embodiment, the biological sample is serum orof a tumor.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, etc. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is by itself or when combined with another composition(s)effective for treating, preventing and/or diagnosing the condition andmay have a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). At least one active agent in thecomposition is an antibody. The label or package insert indicates thatthe composition is used for treating the condition of choice, such ascancer. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises a KLβ antagonist (such as an anti-KLβ antibody); and (b) asecond container with a composition contained therein. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the first and second antibodycompositions can be used to treat a particular condition, e.g. cancer.Alternatively, or additionally, the article of manufacture may furthercomprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

The following are examples of the methods and compositions of theinvention. It is understood that various other embodiments may bepracticed, given the general description provided above.

EXAMPLES

The following materials and methods were used in Examples 1-15.

DNA Constructs:

Full Length Human Klotho Beta Construct:

Total RNA from HepG2 hepatocellular carcinoma cell line was extractedusing RNeasy kit (Quiagen). The human Klotho beta (KLβ) was cloned byreverse transcriptase PCR (RT-PCR) using the SuperScript III One-StepRT-PCR kit (Invitrogen) and the following primers:

Forward primer (SEQ ID NO: 3) 5′-CGGGCGCTAGCATGAAGCCAGGCTGTGCGGCAGG-3′Reverse primer (SEQ ID NO: 4)5′-CAGTGGATCCTTACTTATCGTCGTCATCCTTGTAATCGCTAACAACT CTCTTGCCTTTCTTTC-3′

The resulting KLβ PCR product was digested with NheI and BamHI andligated into pIRESpuro3 (Clontech) to obtain the full-length human KLβc-terminal flag tagged construct (pCMVhKLβ-Flag (SEQ ID NO: 49)).

Full Length Human FGFR4 Construct:

Total RNA from HepG2 hepatocellular carcinoma cell line was extractedusing RNeasy kit (Quiagen). The human FGFR4 cDNA was cloned by reversetranscriptase PCR (RT-PCR) using the SuperScriptIII One-Step RT-PCR kit(Invitrogen) and the following primers:

Forward primer (SEQ ID NO: 5)5′-CCGCCGGATATCATGCGGCTGCTGCTGGCCCTGTTGG-3′ Reverse primer(SEQ ID NO: 6) 5′-CCGCCGGAATTCTGTCTGCACCCCAGACCCGAAGGGG-3′

The resulting FGFR4 PCR product was digested with EcoRV and EcoRI andligated into pIRESpuro3 (Clontech) to obtain the full-length humanFGFR4.

Construct Human FGFR4 with C-Terminal Flag Tag:

The C terminal flag tag was added into human pIRESpuro3FGFR4 usingStratagene XL QuickChange Site-Direct Mutagenesis kit and the followingprimers:

(SEQ ID NO: 7) Forward primer: 5′-GGT CTG GGG TGC AGA CAG GTA AGCCTA TCC CTA ACC CTC TCC TCG GTC TCG ATT CTA CGTAGG AAT TCG GAT CCG CGG C-3′ (SEQ ID NO: 8)Reverse primer: 5′-GCC GCG GAT CCG AAT TCC TAC GTAGAA TCG AGA CCG AGG AGA GGG TTA GGG ATA GGC TTACCT GTC TGC ACC CCA GAC C-3′

Construct Human Secreted KLβ with C-Terminal His Tag:

The human secreted KLβ extracellular domain was obtained by PCR usingpCMVHuKLβ-Flag as the template and the following primers:

(SEQ ID NO: 9) Forward primer: 5′-GAA TTC CAC CAT GAA GCC AGG CTGTGC GGC AGG ATC TCC AG-3′ (SEQ ID NO: 10)Reverse primer: 5′-GGC GCG CCG ACA AGG AAT AAG CAG ACA GTG CAC TCT G-3′

The resulting secreted PCR product was digested with EcoRI and AscI andligated into pRK5_c-His (DNA540910) to obtain pRK5HuKLβΔTM-His (SEQ IDNO:50).

Construct Human KLβ E416A and E693A:

The human KLβ E416A c-terminal flag construct (pCMVhKLβ-Flag E416A (SEQID NO:51)) was obtained by mutation of E416 to A416 in pCMVHuKLβ_Flagusing the XL QuickChange Site-Direct Mutagenesis kit (Stratagene) andthe following primers:

(SEQ ID NO: 11) Forward primer: 5′-CCC TCG AAT CTT GAT TGC TGC GAATGG CTG GTT CAC AGA CAG-3′ (SEQ ID NO: 12)Reverse primer: 5′-CTG TCT GTG AAC CAG CCA TTC GCAGCA ATC AAG ATT CGA GGG-3′

The human KLβ E693A c-terminal flag construct (pCMVhKLβ-Flag E693A (SEQID NO:52)) was obtained by mutation of E693 to A693 in pCMVHuKLβ_Flagusing the XL QuickChange Site-Direct Mutagenesis kit (Stratagene) andthe following primers:

(SEQ ID NO: 13) Forward primer: 5′-GCT CTG GAT CAC CAT CAA CGC GCCTAA CCG GCT AAG TGA C-3′ (SEQ ID NO: 14)Reverse primer: 5′-GTC ACT TAG CCG GTT AGG CGC GTTGAT GGT GAT CCA GAG C-3′hKLβΔTM Conditioned Media

HEK 293 cells were transfected with an empty or a C-terminal his-taggedhuman Klotho beta extracellular domain (hKLβΔTM) containing expressionvector. After transfection the cells were maintained in serum free PS25media for 72-96 hours. The resulting media was filtered, supplemented to40 mM HEPES pH 7.2, concentrated 4 fold and evaluated for hKLβΔTMcontent by Western blot using a monoclonal antibody that binds hKLβ (R&DSystems, catalog no. MAB3738)

Coprecipitation

The concentrated control or hKLβΔTM conditioned medium were supplementedwith Triton-X100 (Calbiochem) to a final concentration of 0.5% andincubated with or without 0.5 μg/ml FGFR-IgG (R&D Systems, catalognumbers as follows: FGFR1 alpha Mb, 655-FR-050; FGFR1 alpha III,658-FR-050; FGFR1 beta Mb, 765-FR-050; FGFR1 beta Inc, 661-FR-050; FGFR2alpha III, 663-FR-050; FGFR2 alpha Inc, 712-FR-050; FGFR2 beta Mb,665-FR-050; FGFR2 beta Inc, 684-FR-050; FGFR3 Mb, 1264-FR-050; FGFR3IIIc, 766-FR-050; FGFR4, 685-FR-050), 0.5 μg/ml heparin (Sigma), 1 μg/mlFGF19 (R&D Systems), 10 μl EZ view Red Protein A affinity gel (Sigma) at4° C. for 18 h. The affinity matrix was centrifuged and washed threetimes with PBS/0.5% Triton-X100 and once with PBS. The pellet was elutedwith SDS-PAGE sample buffer containing 5% β-mercapto ethanol andanalyzed by Western blot using an anti-Klothoβ monoclonal antibody (R&DSystems catalog no. MAB3738), an FGF19 antibody (clone 1A6; GenentechInc.), an anti-FGFR4 antibody (clone 8G11; Genentech Inc.) or a HRPconjugated anti-human IgG antibody (Jackson Immunochemical).

Cell Culture and Stable Cell Lines

HEK 293 (ATCC Accession No. CRL-1573), HepG2 (ATCC Accession No.HB-8065) and Hep 3B (ATCC Accession No. HB-8064) cells were obtainedfrom American Type Culture Collection and maintained in F-12:DMEM mix(50:50) supplemented with 10% fetal bovine serum (FBS) and 2 mML-glutamine. HEK 293 cells stably expressing empty vector, humanfibroblast growth factor receptor 4 (hFGFR4) R388-V5, hFGFR4 G388-V5,human Klotho b-FLAG (hKLβ-FLAG), hFGFR4 R388-V5 and hKLβ-FLAG, or hFGFR4R388-V5 and hKLβ-FLAG were created and grown in selective mediumcontaining 500 μg/ml geneticin and 2.5 μg/ml puromycin.

Time Course of Gene Expression in FGF19 Treated Cells

Cells were plated at 10⁶ cells/well in a 6-well plate and grownovernight in complete media. Cells were washed twice with PBS once withserum free medium and maintained overnight in serum free media. The nextday cells were treated with 20 ng/ml FGF19 for 1, 2, 4, 6, or 24 hoursand at the end of treatment the RNA was extracted using the RNeasy kit(Qiagen). The relative expression level of c-fos, c-jun, junB and KLβwas determined by Taqman.

Semi-Quantitative RT-PCR

Total RNA was extracted using RNeasy kit (Quiagen). Specific primers andfluorogenic probes were used to amplify and quantitate gene expression.The gene specific signals were normalized to the RPL19 housekeepinggene. Triplicate sets of data were averaged for each condition. AllTaqMan RT-PCR reagents were purchased from Applied Biosystems (FosterCity, Calif.). Data are presented as mean+/−SEM. Taqman Primers andprobes (report dye was FAM and quencher dye was TAMRA). Primer sequenceswere as follows:

(SEQ ID NO: 15) RPL19 forward primer: AGC GGA TTC TCA TGG AAC A(SEQ ID NO: 16) RPL19 reverse primer: CTG GTC AGC CAG GAG CTT(SEQ ID NO: 17) RPL19 probe: TCC ACA AGC TGA AGG CAG ACA AGG(SEQ ID NO: 18) hKLβ forward primer: GCA GTC AGA CCC AAG AAA ATA CAG A(SEQ ID NO: 19) hKLβ reverse primer: CCC AGG AAT ATC AGT GGT TTC TTC(SEQ ID NO: 20) hKLβ reverse probe: TGC ACT GTC TGC TTA TTC CTT GT(SEQ ID NO: 21) hc-fos forward primer: CGA GCC CTT TGA TGA CTT CCT(SEQ ID NO: 22) hc-fos reverse primer: GGA GCG GGC TGT CTC AGA(SEQ ID NO: 23) hc-fos probe: CCC AGC ATC ATC CAG GCC CAG(SEQ ID NO: 24) hjunb forward primer: AGT CCT TCC ACC TCG ACG TTT(SEQ ID NO: 25) hjunb reverse primer: AAT CGA GTC TGT TTC CAG CAG AA(SEQ ID NO: 26) hjunb probe: AGC CCC CCC TTC CAC TTT TT (SEQ ID NO: 27)hc-jun forward primer: CGT TAA CAG TGG GTG CCA ACT (SEQ ID NO: 28)hc-jun reverse primer: CCC GAC GGT CTC TCT TCA AA (SEQ ID NO: 29)hc-jun probe: ATG CTA ACG CAG CAG TTG CAA ACA (SEQ ID NO: 30) mKLβforward: TGT GGT GAG CGA AGG ACT GA (SEQ ID NO: 31) mKLβreverse: GGA GTG GGT TGG GTG GTA CA (SEQ ID NO: 32) mKLβprobe: CTG GGC GTC TTC CCC ATG G (SEQ ID NO: 33)mc-fos forward: CCT GCC CCT TCT CAA CGA (SEQ ID NO: 34)mc-fos reverse: TCC ACG TTG CTG ATG CTC TT (SEQ ID NO: 35)mc-fos probe: CCA AGC CAT CCT TGG AGC CAG T (SEQ ID NO: 36)mFGFR4 forward: CGC CAG CCT GTC ACT ATA CAA A (SEQ ID NO: 37)mFGFR4 reverse: CCA GAG GAC CTC GAC TCC AA (SEQ ID NO: 38)mFGFR4 probe: CGT TTC CCT TTG GCC CGA CAG TTC TsiRNA Transfection in HEPG2 and HEP3B Cells:

KLβ and GAPDH siRNA oligos were obtained from Dharmacon.

KLβ siRNA: (SEQ ID NO: 39) Duplex1: Sense: 5′-GCACACUACUACAAACAGAUU-3′(SEQ ID NO: 40) Anti-sense: 5′-UCUGUUUGUAGUAGUGUGCUU-3′ (SEQ ID NO: 41)Duplex2: Sense: 5′-GCACGAAUGGUUCCAGUGAUU-3′ (SEQ ID NO: 42)Anti-sense: 5′-UCACUGGAACCAUUCGUGCUU-3′ (SEQ ID NO: 43)Duplex3: Sense: 5′-CGAUGGAUAUAUUCAAAUGUU-3′ (SEQ ID NO: 44)Anti-sense: 5′-CAUUUGAAUAUAUCCAUCGUU-3′ (SEQ ID NO: 45)Duplex4: Sense: 5′-UGAAAUAACCACACGCUAUUU-3′ (SEQ ID NO: 46)Anti-sense: 5′-AUAGCGUGUGGUUAUUUCAUU-3′ (SEQ ID NO: 47)GAPDH siRNA: Sense: 5′-UGGUUUACAUGUUCCAAUA-3′ (SEQ ID NO: 48)Antisense: 5′ -UAUUGGAACAUGUAAACCA-3′

The various siRNA duplex were transfected using the DharmaFECTtransfection kit (Dharmacon) and following the manufacturer'srecommended protocol. Twenty-four hours post-transfection, the cellswere washed twice with PBS and once with serum free media and maintainedin serum free media overnight. The following days the cells were treatedwith 20 ng/ml FGF19 (R&D Systems) for 2 hours. The RNA samples wereprepared with a RNeasy kit (Qiagen). The relative levels of c-fos,RPL19, and KLβ expression were determined by Taqman.

In Vitro KLβ Antibody Treatment

HEPG2 cells were plated at 10⁶ cells/well in a 6-well plate and grownovernight in complete media. Cells were washed 3 times with serum freemedia containing 0.1% BSA and maintained in the same media overnight.The next day the cells were treated with 10 μg/ml KLβ specificpolyclonal antibody (R&D Systems; cat# AF2619) or a control antibody for4 h. The cells were then treated with 100 ng/ml FGF19 for 2 hours andthe RNA was extracted using the RNeasy kit (Qiagen). The relativeexpression level of c-fos was determined by Taqman.

Co-Immunoprecipitation

HEK 293 cells transiently (24-48 hour transfection) or stably expressingempty vector, hFGFR4 R388-V5, hKLβ-FLAG, or hFGFR4 R388-V5 and hKLβ-FLAGwere lysed with RIPA lysis buffer (PBS containing 1% Triton X-100 and 1%NP-40) supplemented with Complete EDTA-free protease inhibitor cocktail(Roche). Total protein concentrations were determined by BCA proteinassay (Pierce). Equal amounts of total protein for each sample lysatewere immunoprecipitated with EZview Red anti-FLAG M2 affinity gel(Sigma) or anti-V5 agarose affinity gel (Sigma) at 4° C. overnight.Immunoprecipitated proteins were washed three times with TBST and thenincubated without or with 1 mg/ml FGF-19 (R&D Systems) in F-12:DMEM mix(50:50) supplemented with 0.5% FBS and 0.5% Triton X-100 at 4° C. for 3hours. The beads were then washed once with Krebs-Ringer-HEPES (KRH)buffer containing 1% Triton X-100 and three times with KRH buffer.Immunoprecipitated proteins were eluted by addition of 1× NuPAGE LDSsample buffer (Invitrogen) and boiling for 5 minutes. Eluted proteins insample buffer were recovered and 1× NuPAGE reducing agent (Invitrogen)was added and then boiled for 10 minutes. Protein samples wereelectrophoresed on 4-12% NuPAGE Bis-Tris gels followed by transfer tonitrocellulose membranes and subjected to subsequent immunoblot analysesusing anti-hKLβ antibody (1 mg/ml, R&D Systems; catalog no. MAB3738),anti-hFGFR4 antibody (1 mg/ml, clone 8G1, Genentech), or anti-FGF-19antibody (1 mg/ml, clone 1A6, Genentech). Signal was detected using ECLPlus substrate (GE Healthcare).

FGF Pathway Activation

HEK 293 cells transiently (24 hour transfection) or stably expressingempty vector, hFGFR4 R388-V5, hFGFR4 G388-V5, hKLβ-FLAG, hFGFR4 R388-V5and hKLβ-FLAG, or hFGFR4 G388-V5 and hKLβ-FLAG were treated with 0, 1,10, or 100 ng/ml of FGF-19 (R&D Systems), 20 ng/ml of FGF-1 (FGF acidic,R&D Systems), or 20 ng/ml epidermal growth factor (Roche) for 10minutes. Cells were lysed with RIPA lysis buffer (Upstate) supplementedwith Complete EDTA-free protease inhibitor cocktail (Roche) andphosphatase inhibitor cocktails 1 and 2 (Sigma). Total proteinconcentrations were determined by BCA protein assay (Pierce). Foranalysis of FRS2 and ERK1/2 phosphorylation, equal amounts of totalprotein were electrophoresed on 10% NuPAGE Bis-Tris gels (Invitrogen)followed by transfer to nitrocellulose membranes and subsequentimmunoblot analyses using anti-phospho-FRS2 antibody (1:1,000, CellSignaling Technology) or anti-ERK1/2 antibody (1:1,000, Cell SignalingTechnology). For detection of total FRS2 and ERK1/2, membranes werestripped and reprobed with anti-FRS2 antibody (1 mg/ml, UpstateBiotechnology) or anti-ERK1/2 antibody (1:1,000, Cell SignalingTechnology). Signal was detected using ECL Plus substrate (GEHealthcare).

In Vivo Experiments

All animal protocols were approved by an Institutional Animal Care andUse Committee. Five- to six-week-old Female FVB mice were obtained fromCharles River Laboratories. The mice were provided standard feed andwater ad libitum until 12 hours prior to treatment at which time feedwas removed. Mice were injected intravenously with vehicle (PBS) or with1 mg/kg FGF19. When indicated, mice were injected intravenously with 2.2mg/kg KLβ antibody (R&D Systems; cat# AF2619) 3, 9 or 24 hours beforethe intravenous FGF19 inoculation. After 30 min, mice from all groupswere sacrificed and tissue samples were collected, frozen in liquidnitrogen, and stored at −70° C. Total RNA from frozen tissue samples wasprepared using the RNAeasy kit (Qiagen). Groups of 3-5 animals wereanalyzed for each condition. Data are presented as the mean±SEM and wereanalyzed by the Student t-test.

In Silico Expression Analysis

For KLβ and FGFR4 expression analysis, plots are based on normalizedgene expression data extracted from the BioExpress™ database (GeneLogic, Inc., Gaithersburg, Md., USA). The KLβ expression reported herecorresponds to the signal given by the probe number 244276_at and204579_at, respectively, in human tissues analyzed on AffymetrixGeneChips. The bold center line indicates the median; the box (white,normal; gray, tumor) represents the interquartile range between thefirst and third quartiles. The distribution of the values for a givensamples is indicated by broken lines. The human sample collection hasbeen described by the originator of the BioExpress™ database (Shen-Ong GL et al. Cancer Res 2003; 63: 3296-301. The respective hybridizationswere performed on Affymetrix HG-U133P oligonucleotide chips (Affymetrix,Inc., Santa Clara, Calif., USA): Briefly, these chips are based on25-mer oligonucleotides and allow the detection of more than 33,000well-substantiated human genes, with probe sets of 11 oligonucleotidesused per transcript.

Example 1 KLβ Extracellular Domain and FGF19 Binding Specificities areRestricted to FGFR4

FGF19, heparin and FGFRs-Fc fusion proteins were incubated inconditioned medium containing KLβΔTM. The protein interactions were thenanalyzed by co-precipitation. KLβ and FGF19 co-associated only withFGFR4 and were pulled down only with FGFR4-Fc (FIG. 1A). These dataindicated that the binding specificities of KLβ extracellular domain andFGF19 are restricted to FGFR4 and that KLβ extracellular domain, FGF19and FGFR4 likely form a tripartite complex.

Example 2 KLβ Binding to FGFR4 is Promoted by FGF19 and Heparin

To evaluate the contribution of each component to complex formation, theco-precipitation assay was used. Control or KLβΔTM containingconditioned medium was incubated in the presence or the absence ofFGFR4-Fc, FGF19, and heparin. In the absence of heparin and FGF19, nointeraction was detected between KLβ and FGFR4-Fc (FIG. 1B). Heparin wasa weak promoter, whereas FGF19 was a strong promoter of the KLβ-FGFR4interaction. The maximal level of stabilization of the KLβ-FGFR4-Fcinteraction occurred in the presence of both heparin and FGF19.Conversely, FGF19 binding to FGFR4-Fc required the presence of heparinor KLβ. The maximal level of FGF19 binding to FGFR4 occurred when bothheparin and KLβ were included in the reaction. These data demonstratethat KLβ is sufficient to support FGF19 binding to FGFR4. It also showsthat KLβ promotes the previously demonstrated, heparin-dependentinteraction of FGF19 with FGFR4 (Xie et al (1999) Cytokine 11:729-35).Therefore, each individual component contributes to the stability of theFGF19-FGFR4-KLβ-heparin complex.

Compared with the paracrine FGF family members, FGF19 has a lowheparin-binding affinity that allows it to act in an endocrine fashionwithout being tethered to the pericellular proteoglycan of the secretingcells (Choi, M et al (2006) Nat Med 12: 1253-5; Harmer, N J et al (2004)Biochem 43:629-40; Inagaki, Y et al (2005) Cell Metab 2:217-25;Lundasen, T et al (2006) J Intern Med 260:530-6). The topology of theFGF19 heparin-binding site prevents FGF19 from forming hydrogen bondswith heparin when FGF19 is bound to its receptor Goetz, R et al (2007)Mol Cell Biol. 27:3417-28). Therefore KLβ may act as an FGFR4co-receptor that stabilizes the weak FGF19-FGFR4-heparin interaction.

Example 3 Interaction of Transfected FGFR4 and KLβ at the Cell Surface

To test whether KLβ and FGFR4 also participate in the formation of acomplex with FGF19 and heparin at the cell surface we evaluated theability of FGFR4 and KLβ to immunoprecipitate FGF19 from lysates oftransiently or stably transfected cells in the presence or the absenceof heparin. No detectable FGF19 was co-precipitated from lysates ofcells transfected with only a control or an FGFR4-expression vector(FIGS. 1 C and 1D). FGFR4 and KLβ pulled down FGF19 from KLβ-transfectedcell lysate only in the presence of heparin indicating that KLβtransfection promotes heparin-dependent FGF19 binding to the endogenousHEK293 FGFR4.

These findings suggest that FGFR4 and KLβ exist as a preformed complexand that their interaction is not enhanced by FGF19.

Example 4 Interaction of Endogenous FGFR4 and KLβ at the Cell Surface

The KLβ and FGFR4 transmembrane domains could directly interact witheach other or promote the interaction of the proteins by tethering themto the cell surface. To test the hypothesis that KLβ and FGFR4 form aconstitutive complex at the cell surface, we evaluated whether KLβco-immunoprecipitated with FGFR4 from HEPG2 cell lysates in the absenceof FGF or heparin. Incubation of HEPG2 cell lysates with an antibodyagainst FGFR4 immunoprecipitated FGFR4 and KLB, whereas no protein wasimmunoprecipitated with the control antibody (FIG. 1E), showing that theendogenous transmembrane KLβ and FGFR4 form a constitutive heparin- andligand-independent complex. The KLβ-FGFR4 cell surface complex mightalter the heparin- and ligand-induced receptor dimerization that waspreviously described for paracrine FGFs (Plonikov, A (1999) Cell98:641-50; Schlessinger, J et al (2000) Mol Cell 6:743-50). These dataindicate that endogenous KLβ and FGFR4 interact at the cell surface.Applicants note that FIG. 4 of Applicant's prior application, U.S. Ser.No. 60/909,699, filed Apr. 2, 2007, (corresponding to FIG. 1E of thepresent application) shows a molecular weight mark at 150 kDa, whileFIG. 1E shows a 130 kDa molecular weight mark. The mislabeling of FIG. 4of the '699 application was an inadvertent obvious error, as KLβ isknown to be a 130-kDa protein (see, e.g., Ito et al., Mech. Dev. 98(2000) 115-119).

Example 5 FGF19 Binding to FGFR4 and KLβ at the Cell Surface

To test whether KLβ and FGFR4 also participate in the formation of acomplex with FGF19 and heparin at the cell surface we evaluated theability of FGFR4 and KLβ to immunoprecipitate FGF19 from lysates oftransiently or stably transfected cells in the presence or the absenceof heparin. No detectable FGF19 was co-precipitated from lysates ofcells transfected with only a control or an FGFR4-expression vector(FIGS. 1 C and 1D). FGFR4 and KLβ pulled down FGF19 from KLB-transfectedcell lysate only in the presence of heparin indicating that KLβtransfection promotes heparin-dependent FGF19 binding to the endogenousHEK293 FGFR4.

In lysates from of KLβ- and FGFR4-co-transfected cells, KLβ and FGFR4readily pulled down FGF19. This interaction was further stabilized inthe presence of heparin (FIGS. 1 C and 1D). These data show that KLβ isrequired for FGF19 binding to the cell surface FGFR4 and that heparinpromotes this interaction. In addition, FGFR4 and KLβ readily interactedin a heparin- and ligand-independent manner in co-transfected cells.This result contrasts with the heparin- and ligand-dependent complexformation observed with the secreted chimeric FGFR4 and KLβ proteins.This discrepancy indicates a role for the KLβ and FGFR4 transmembranedomains in the complex formation.

These findings suggest that FGF19 binds to KLβ and FGFR4 at the cellsurface and that heparin enhances this interaction.

Example 6 FGF19 Represses Klotho Beta Expression

The effect of FGF19 on KLβ expression in various cell lines wasevaluated. We detected high KLβ expression in liver cell lines (HepG2and Hep3B) but only traces in kidney (HEK293) or colon cell lines (SW620and Colo205; FIG. 3A). Upon exposure to FGF19, KLβ expression in HepG2and Hep3B was gradually repressed, to 50-60% the level of unexposedcells after 6 hours, and remained at this level for at least 24 hours.Exposure to FGF19 did not affect KLβ expression levels in the other celllines. The repression of KLβ expression by FGF19 might be a regulatorynegative feedback mechanism in liver cells.

Example 7 FGF19 is Required for FGF19 Downstream Modulation of GeneExpression

Because a plethora of physiological and pathological stimuli induce thegenes of the Fos and Jun family in a wide variety of cell types wetested whether FGF19 modulates c-Fos, JunB, and c-Jun expression invarious cell lines (Ashida, R et al (2005) Inflammapharmacology 13:113-25; Hess, J et al (2004) Biochemistry 43:629-40; Shaulian, E et al(2002) Nat Cell Biol 4:E131-6). FGF19 upregulated c-Fos and JunBexpression, as well as c-Jun expression to a lesser extent, inKLβ-expressing cells (HepG2 and HEP3B; FIG. 3 B-D). The induction c-Fos,JunB and c-Jun expression occurred within 30 minutes of exposure toFGF19 and in most cases expression returned to basal levels after 6hours. JunB expression remained elevated for at least 24 hours in HEP3Bcells (FIG. 3C).

These data indicate that FGF19 induced c-Fos, Junb and c-Jun expressiononly in KLβ expressing cells.

Example 8 KLβ Knock-Down Inhibits FGF19-Dependent c-Fos Induction

To test whether KLβ promotes FGF19 signaling and c-Fos induction inHEP3B and HEPG2 cells, we inhibited KLβ expression using specificsiRNAs. KLβ siRNA transfection significantly reduced KLβ mRNA andprotein expression in HEP3B (FIGS. 3G and E) and HEPG2 cells (FIG. 5A).The individual transfection of four different KLβ siRNAs significantlyattenuated FGF19-mediated FRS2 and ERK1/2 phosphorylation (FIG. 3F, 5B).In addition, transfection of HEP3B cells with KLβ siRNA transfectioninhibited FGF19 mediated c-Fos induction by 62%-80%, compared to thecontrol cells (FIG. 3G). Similarly, transfection of HEPG2 cells with KLβsiRNA reduced the levels of FGF19-dependent FRS2 and ERK1/2phosphorylation as well as c-Fos induction as compared with the controlcells (FIG. 5C). These results indicate that KLβ expression is requiredfor FGF19-dependent pathway activation and c-Fos induction. Theseresults indicate that KLβ expression is required for FGF19-dependentc-Fos induction.

To further assess the participation of KLβ in FGF19-mediated c-Fosinduction, we transfected HEK293 cells with empty, KLβ-, FGFR4-, or acombination of KLβ- and FGFR4-expression vectors and exposed the cellsto FGF19. Only cells transfected with both KLβ and FGFR4 expressionvectors induced c-Fos in response to FGF19 (FIG. 3H). These dataindicate that KLβ is required for FGF19 pathway activation andmodulation of gene regulation.

Example 9 Treatment with an Anti-KLβ Antibody Inhibits FGF19-Dependentc-Fos Induction

To further evaluate the contribution of KLβ to the FGF19-dependent c-Fosinduction, HEPG2 cells were treated with an anti-KLβ antibody (raisedagainst mouse KLβ, but cross-reactive with human KLβ) or a controlantibody before treatment of the cells with FGF19. Anti-KLβ antibodytreatment reduced FGF19-dependent c-Fos induction by 80% whereas thecontrol antibody did not show any significant effect (FIG. 6).

These results demonstrate that targeting KLβ with a specific antibodyinhibits FGF19 activity.

Example 10 KLβ is Required for FGF19 Signaling

To test whether KLβ contributes to the activation of the FGF19 signalingpathway, we evaluated the effects of FGF19 on FGFR substrate 2 (FRS2)and extracellular-signal regulated kinase-1 and -2 (ERK1/2)phosphorylation in KLβ- and/or FGFR4-transfected HEK 293 cells, as wellas controls. FGF19 did not promote FRS2 or ERK1/2 phosphorylation incells transfected with an empty expression vector (FIG. 2). HEK 293cells transfected with KLβ or FGFR4 only showed a weak, dose-dependentincrease in ERK1/2 phosphorylation but no detectable FRS2phosphorylation following exposure to FGF19. The co-transfection ofFGFR4 with KLB promoted FGF19 signaling in HEK 293 cells, indicated bythe robust, dose-dependent increase of both FRS2 and ERK1/2phosphorylation. One possible explanation for this effect is that local,high concentrations of FGF19 and FGFR4 allow for weak signaling in theabsence of KLβ. However, because FGF19 has an endocrine function and itsaverage circulating concentration is 193±36 pg/mL (range of 49-590pg/mL), this is unlikely (Lundasen, T et al (J Intern Med 260:530-6).Therefore KLβ's robust induction of FGF19 signaling is likely to occurat physiological concentrations of FGF19.

Example 11 KLβ Active Site Mutation Inhibits FGF19 Pathway Activation

The requirement of KLβ for FGF19-stimulated activity was assessed bydetection of downstream signaling (i.e. phospho-FRS2 and -ERK1/2) in HEK293 cells transfected with wild-type (wt) KLβ or KLβ mutants. WtKLβ-transfected cells showed detectable phospho-FRS2 and phospho-ERK1/2upon treatment with 100 ng/ml FGF19 (FIG. 7). When treated with the sameFGF19 dose, the KLβ E416A mutant (containing a glutamate to alaninemutation in one of the putative active sites; see Ito et al. (2000)Mech. Dev. 98 (1-2):115-119 for a description of this residue) did notshow detectable phosphorylation of either FRS2 or ERK1/2. Thus, amutation in the E416 putative active site of KLβ eliminated FGFR4downstream signaling, suggesting that KLβ enzymatic activity is requiredfor FGFR4 signaling.

FGF19 treatment of cells transfected with the KLβ E693A mutant((containing a glutamate to alanine mutation in one of the putativeactive sites; see Ito et al., supra, for a description of this residue)showed similar levels of phosphorylation of phospho-FRS2 orphospo-ERK1/2 to FGF19-treated cells expressing wt KLβ (FIG. 7). Thus, amutation in the E693 putative active site of KLβ did not affect KLβactivity. Therefore only the E416 putative active site of KLβ isrequired for the FGF19 dependent stimulation of FRS2 and ERK1/2phosphorylation. KLβ protein expression was detected to demonstrate thatcells transfected with vectors expressing wt or mutant KLβ expressedequivalent amounts of protein.

These findings corroborate the finding that FGF19 signaling throughFGFR4 is enhanced by the presence of KLβ and further suggest that KLβenzymatic activity is required for FGFR4 signaling.

Example 12 Distribution of KLβ Expression in Mouse Tissues In Vivo

To test the hypothesis that FGF19 acts only on tissues that express bothFGFR4 and KLβ, we first surveyed KLβ and FGFR4 distribution in variousmouse organs using semi-quantitative RT-PCR. The relative mRNA levelsrepresent the relative fold expression, compared with brain (organ withthe lowest expression surveyed). KLβ was predominantly expressed inliver (FIG. 4C). Lower levels of KLβ expression were also found inadipose and colon. Additional organs tested showed marginal expressionlevels of KLβ. FGFR4 was highly expressed in liver, lung, adrenals,kidney and colon (FIG. 4D). Lower levels of FGFR4 expression were alsoobserved in intestine, ovaries, muscle and pancreas. The overall KLβ andFGFR4 distribution in mouse tissues was similar that of human tissues.However, contrary to the findings in human tissues, no consistent KLβ orFGFR4 expression could be detected in mouse pancreas. In addition, a lowlevel of KLβ expression was detected in mouse colon, whereas noexpression was found in the corresponding human tissues. Thesedifferences might be attributable to species- and/or strain-specifictissue distribution. These data indicate that liver is the only mouseorgan in which KLβ and FGFR4 are highly co-expressed.

Example 13 FGF19 Acts Specifically on Mouse Liver

To determine the FGF19 specific site of action, we compared the levelsof c-Fos expression in organs of mice injected with FGF19 with those ofmice injected with PBS (controls). We chose to monitor the c-Fosresponse to FGF19 because c-Fos expression is ubiquitous and itsinduction is sensitive to FGF19 stimulation. C-Fos expression was1300-fold higher in the livers of mice injected with FGF19 compared withthe livers of mice injected with PBS (FIG. 4E). The FGF19-dependentc-Fos induction was at least 150-fold lower in all other organs tested.The activity of FGF19 in liver was confirmed by a 98% inhibition ofCYP7A1 expression (FIG. 4F). These data demonstrate that FGF19 actsspecifically in liver, the only mouse organ that expresses high levelsof both KLβ and FGFR4.

Together, the data shown in Examples 12 and 13 demonstrate that FGF19requires KLβ for binding to FGFR4, intracellular signaling, anddownstream gene modulation. Most importantly, the requirement for KLβrestricts the endocrine activity of FGF19 to tissues that express bothFGFR4 and KLβ. The liver-specific activity of FGF19 is supported by thismolecular mechanism. These data demonstrate that the liver is a majorsite of action of FGF19 in the mouse.

Example 14 Treatment with Anti-KLβ Antibody In Vivo InhibitsFGF19-Dependent c-Fos Induction in Mouse Liver

To evaluate the KLβ requirement for FGF19 activity in vivo,FGF19-dependent c-Fos induction was determined in liver of mouse treatedwith a KLβ antibody for different lengths of time. Treatment of micewith 2.5 mg/kg of KLβ antibody 3, 9 or 24 hours before a FGF19 injectionreduced the liver specific FGF19-mediated c-Fos induction by 58%, 68%and 91% respectively (FIG. 8).

These data indicate that KLβ is required for FGF19 signaling throughFGFR4 in vivo. In addition, the data further demonstrate thatKLβ-specific antibodies can be used to inhibit FGF19 activity in vivo.

Example 15 Analysis of KLβ and FGFR4 Expression in Normal and CancerTissues

KLβ and FGFR4 expression were evaluated in a variety of human tissues byanalyzing the BioExpress database (Gene Logic, Inc., Gaithersburg, Md.,USA). In decreasing order of signal intensity, KLβ was expressed inadipose, liver, pancreas, and breast tissues (FIG. 4A). In decreasingorder of signal intensity, FGFR4 was expressed in liver, lung, gallbladder, small intestine, pancreas, colon, lymphoid, ovary and breasttissues (FIG. 4B). These data show that KLβ expression is restricted toonly a few tissues, whereas FGFR4 expression is more widely distributed.A high level of co-expression of KLβ and FGFR4 was observed only inliver and pancreas. Because the expression of KLβ and FGFR4 are requiredfor FGF19 activity, this finding suggests that liver and pancreas arethe major organs in which they are active. Marginal levels of KLβ andFGFR4 expression were also observed in breast tissues. KLβ was highlyexpressed in adipose tissues but the absence of FGFR4 precludes thefunction of FGF19 in this tissue. It is possible that KLβ promotes theactivity of other endocrine FGF family members with different FGFRbinding specificity in adipose tissues. Notably, FGF21 regulates glucoseuptake by acting specifically on adipose tissue by an endocrinemechanism (Kharitonenkov, A, et al (2005) J Clin Invest 115:1627-35).Because of its low heparin-binding affinity, FGF21 might require KLβ tosignal through FGFR1 and FGFR2 (Goetz, R et al (2007) Mol Cell Biol27:3417-28; Kharitonenkov, supra).

KLβ expression and FGFR4 were also evaluated in a variety of cancertissues. KLβ expression is generally reduced in other cancer tissuescompared to the relevant normal tissue (FIG. 9). In decreasing order ofsignal intensity, FGFR4 is expressed in the following cancer tissues:liver, colon, stomach, esophagus, kidney, testis, small intestine,pancreas, ovary and breast (FIG. 10). These data show that FGFR4expression is widely distributed and that its normal expression isaltered in cancer.

Example 16 Discussion

In this study we have provided evidence that FGF19 requires KLB forbinding to FGFR4, intracellular signaling, and down-stream genemodulation. However, the reason for such a requirement is still unclear.Compared with the paracrine FGF family members, FGF19 has a lowheparin-binding affinity that allows it to act in an endocrine fashionwithout being tethered to the pericellular proteoglycan of the secretingcells (3, 6, 8, 15). The topology of the FGF19 heparin-binding siteprevents FGF19 from forming hydrogen bonds with heparin when FGF19 isbound to its receptor (5). Therefore KLB may act as an FGFR4 co-receptorthat stabilizes the weak FGF19-FGFR4-heparin interaction.

In addition, we have shown that FGFR4 and KLβ readily interacted in aheparin- and ligand-independent manner at the cell surface. This resultcontrasts with the heparin- and ligand-dependent complex formationobserved with the secreted chimeric FGFR4 and KLβ proteins. Thisdiscrepancy may indicate a role for the KLβ and FGFR4 transmembranedomains in the complex formation. The KLβ and FGFR4 transmembranedomains could directly interact with each other or promote theinteraction of the proteins by tethering them to the cell surface. TheKLβ-FGFR4 cell surface complex might alter the heparin- andligand-induced receptor dimerization that was described previously forparacrine FGFs (18, 20).

We found high expression of Ku in adipose tissue. However, the absenceof FGFR4 expression precludes FGF19 activity in this tissue. Therefore,it is possible that KLβ promotes the activity of other endocrine FGFfamily members with different FGFR binding specificity in adiposetissues. Notably, KLβ was recently shown to be required for FGF21adipose-specific activity (17). Because of its low heparin-bindingaffinity, FGF21 might require KLβ to signal through FGFR1 and FGFR2 (6,12).

Together, these data demonstrate that FGF19 requires KLβ for binding toFGFR4, intracellular signaling, and downstream gene modulation. Mostimportantly, the requirement for KLβ restricts the endocrine activity ofFGF19 to tissues that express both FGFR4 and KLβ. The liver-specificactivity of FGF19 is supported by this molecular mechanism.

PARTIAL REFERENCE LIST

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Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention.

What is claimed is:
 1. A method for modulating a disorder associatedwith expression or activity of KLβ, the method comprising administeringan effective amount of a KLβ modulator to an individual in need of suchtreatment.
 2. A method for treating a disorder associated withexpression or activity of KLβ, the method comprising administering aneffective amount of a KLβ antagonist to an individual in need of suchtreatment.
 3. The method of claim 2, wherein the disorder is a tumor, acancer, or a cell proliferative disorder.
 4. The method of claim 3,wherein the tumor, cancer, or cell proliferative disorder ishepatocellular carcinoma, pancreatic cancer, non-small cell lung cancer,breast cancer, or colorectal cancer.
 5. The method of claim 2, whereinthe disorder is a liver disorder.
 6. The method of claim 5, wherein theliver disorder is cirrhosis.
 7. The method of claim 2, wherein thedisorder is hypoglycemia, cholestasis or dysregulation of bile acidmetabolism.
 8. The method of claim 2, wherein the disorder is wasting.9. The method of claim 8, wherein the individual further has a tumor, acancer, and/or a cell proliferative disorder.
 10. The method of claim 2,further comprising administering to the subject an effective amount of asecond medicament, wherein the KLβ antagonist is a first medicament 11.The method of claim 10, wherein the second medicament is an antibody, achemotherapeutic agent, a cytotoxic agent, an anti-angiogenic agent, animmunosuppressive agent, a prodrug, a cytokine, a cytokine antagonist,cytotoxic radiotherapy, a corticosteroid, an anti-emetic, a cancervaccine, an analgesic, or a growth-inhibitory agent.
 12. The method ofclaim 11, wherein the second medicament is tamoxifen, letrozole,exemestane, anastrozole, irinotecan, cetuximab, fulvestrant,vinorelbine, erlotinib, bevacizumab, vincristine, imatinib, sorafenib,lapatinib, or trastuzumab.
 13. The method of claim 10, wherein thesecond medicament is administered prior to or subsequent to theadministration of the KLβ antagonist.
 14. The method of claim 10,wherein the second medicament is administered concurrently with the KLβantagonist.
 15. The method of claim 2, wherein the KLβ antagonist is anantibody
 16. The method of claim 15, wherein the antibody is amonoclonal antibody.
 17. The antibody of claim 15, wherein the antibodyis selected from the group consisting of a chimeric antibody, ahumanized antibody, an affinity matured antibody, a human antibody, anda bispecific antibody.
 18. The antibody of claim 15, wherein theantibody is an antibody fragment.
 19. The antibody of claim 15, whereinthe antibody is an immunoconjugate.
 20. The method of claim 2, furtherwherein a biological sample of the individual expresses (i) KLβ, (ii)KLβ and FGF, (iii) KLβ and FGFR, or (iv) KLβ, FGF and FGFR.
 21. Themethod of claim 20, wherein the biological sample expresses KLβ.
 22. Themethod of claim 20, wherein the biological sample expresses KLβ andFGFR4.
 23. The method of claim 20, wherein the biological sampleexpresses KLβ, FGF19 and FGFR4.
 24. The method of claim 1, wherein theKLβ modulator is a KLβ agonist.
 25. The method of claim 24, wherein thedisorder is obesity or an obesity-related condition.
 26. The method ofclaim 25, wherein the obesity-related condition is diabetes mellitus,cardiovascular disease, insulin resistance, hypertension,hypercholesterolemia, thromboembolic disease, atherosclerosis,dyslipidemia, osteoarthritis, gallbladder disease, osteoarthritis, andsleep apnea and other respiratory disorders.
 27. The method of claim 25,wherein the disorder is hyperglycemia.
 28. A method for inducing anincrease in insulin sensitivity, the method comprising administration ofan effective dose of a KLβ agonist to an individual in need of suchtreatment.
 29. A method for reducing total body mass, the methodcomprising administration of an effective dose of a KLβ agonist to anindividual in need of such treatment.
 30. A method for reducing at leastone of triglyceride and free fatty acid levels, the method comprisingadministration of an effective dose of a KLβ agonist to an individual inneed of such treatment.
 31. A method for detection of KLβ, the methodscomprising detecting KLβ in a sample.
 32. The method of claim 31,wherein the sample is a biological sample from a cancer, a tumor or acell proliferative disorder.
 33. The method of claim 32, wherein thebiological sample expresses FGF or FGFR.
 34. A method of identifying acandidate inhibitor substance that inhibits KLβ binding to FGFR, saidmethod comprising: (a) contacting a candidate substance with a firstsample comprising FGFR, FGF and KLβ, and (b) comparing amount of FGFRbiological activity in the sample with amount of FGFR biologicalactivity in a reference sample comprising similar amounts of KLβ, FGFand FGFR as the first sample but that has not been contacted with saidcandidate substance, whereby a decrease in amount of FGFR biologicalactivity in the first sample compared to the reference sample indicatesthat the candidate substance is capable of inhibiting KLβ binding toFGFR.
 35. A method of determining whether a candidate substance promotesKLβ biological activity, said method comprising: (a) contacting acandidate substance with a first sample comprising FGFR and KLβ, and (b)comparing amount of FGFR biological activity in the sample with amountof FGFR biological activity in a reference sample comprising similaramounts of KLβ and FGFR as the first sample but that has not beencontacted with said candidate substance, whereby an increase in amountof FGFR biological activity in the first sample compared to thereference sample indicates that the candidate substance is capable ofpromoting KLβ binding to FGFR.